Methods of Manipulating Phagocytosis Mediated by CD47

ABSTRACT

Methods are provided to manipulate phagocytosis of cancer cells, including e.g. leukemias, solid tumors including carcinomas, etc.

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under contract CA086017awarded by the National Institutes of Health. The Government has certainrights in the invention.

BACKGROUND

The reticuloendothelial system (RES) is a part of the immune system. TheRES consists of the phagocytic cells located in reticular connectivetissue, primarily monocytes and macrophages. The RES consists of 1)circulating monocytes; 2) resident macrophages in the liver, spleen,lymph nodes, thymus, submucosal tissues of the respiratory andalimentary tracts, bone marrow, and connective tissues; and 3)macrophage-like cells including dendritic cells in lymph nodes,Langerhans cells in skin, and microglial cells in the central nervoussystem. These cells accumulate in lymph nodes and the spleen. The RESfunctions to clear pathogens, particulate matter in circulation, andaged or damaged hematopoietic cells.

To eliminate foreign cells or particles in the innate immune response,macrophage-mediated phagocytosis is induced when the phosphatidylserinereceptor (PSR) reacts to phosphatidylserine (PS), which can beexternalized from the membranes of dead cells, such as apoptotic andnecrotic cells. In turn, the interaction between PS and PSR plays acrucial role in the clearance of apoptotic cells by macrophages. Oncephagocytosis has been performed by macrophages, the inflammatoryresponse is downregulated by an increase in factors such as IL-10,TGF-□, and prostaglandin E2 (PGE2). The strict balance between theinflammatory and anti-inflammatory responses in both innate and adaptiveimmunity plays a critical role in maintaining cellular homeostasis andprotecting a host from extrinsic invasion.

The causal relationship between inflammation and the neoplasticprogression is a concept widely accepted. Data now support the conceptof cancer immunosurveillance—that one of the physiologic functions ofthe immune system is to recognize and destroy transformed cells.However, some tumor cells are capable of evading recognition anddestruction by the immune system. Once tumor cells have escaped, theimmune system may participate in their growth, for example by promotingthe vascularization of tumors.

Both adaptive and innate immune cells participate in the surveillanceand the elimination of tumor cells, but monocytes/macrophages may be thefirst line of defense in tumors, as they colonize rapidly and secretecytokines that attract and activate dendritic cells (DC) and NK cells,which in turn can initiate the adaptive immune response againsttransformed cells.

Tumors that escape from the immune machinery can be a consequence ofalterations occurring during the immunosurveillance phase. As anexample, some tumor cells develop deficiencies in antigen processing andpresentation pathways, which facilitate evasion from an adaptive immuneresponse, such as the absence or abnormal functions of components of theIFN-□ receptor signaling pathway. Other tumors suppress the induction ofproinflammatory danger signals, leading, for example, to impaired DCmaturation. Finally, the inhibition of the protective functions of theimmune system may also facilitate tumor escape, such as theoverproduction of the anti-inflammatory cytokines IL-10 and TGF-β, whichcan be produced by many tumor cells themselves but also by macrophagesor T regulatory cells.

A tumor can be viewed as an aberrant organ initiated by a tumorigeniccancer cell that acquired the capacity for indefinite proliferationthrough accumulated mutations. In this view of a tumor as an abnormalorgan, the principles of normal stem cell biology can be applied tobetter understand how tumors develop. Many observations suggest thatanalogies between normal stem cells and tumorigenic cells areappropriate. Both normal stem cells and tumorigenic cells have extensiveproliferative potential and the ability to give rise to new (normal orabnormal) tissues. Both tumors and normal tissues are composed ofheterogeneous combinations of cells, with different phenotypiccharacteristics and different proliferative potentials.

Stem cells are defined as cells that have the ability to perpetuatethemselves through self-renewal and to generate mature cells of aparticular tissue through differentiation. In most tissues, stem cellsare rare. As a result, stem cells must be identified prospectively andpurified carefully in order to study their properties. Perhaps the mostimportant and useful property of stem cells is that of self-renewal.Through this property, striking parallels can be found between stemcells and cancer cells: tumors may often originate from thetransformation of normal stem cells, similar signaling pathways mayregulate self-renewal in stem cells and cancer cells, and cancers maycomprise rare cells with indefinite potential for self-renewal thatdrive tumorigenesis.

Study of cell surface markers specific to or specifically upregulated incancer cells is pivotal in providing targets for reducing growth of orfor depleting cancer cells. Provided herein is a marker for myeloidleukemia, especially a marker for Acute Myeloid Leukemia (AML). Ourstudies have revealed a role of this marker in helping AML stem cellsavoid clearance by phagocytosis. Methods are provided for using thismarker to increase phagocytosis of AML stem cells (AML SCs), as well asto improve transplantation of hematopoietic and progenitor stem cells.

Interestingly, certain markers are shown to be shared by leukemia stemcells and hematopoietic stem cells (HSCs). During normal development,HSCs migrate to ectopic niches in fetal and adult life via the bloodstream. Once in the blood stream, HSCs must navigate the vascular bedsof the spleen and liver before settling in a niche. At these vascularbeds, macrophages function to remove damaged cells and foreign particlesfrom the blood stream. Furthermore, during inflammatory states,macrophages become more phagocytically active. The newly arriving stemcells thus face the possibility of being phagocytosed while en route,unless additional protection can be generated. Exploration of mechanismsby which the endogenous HSC avoid being cleared by phagocytosis canprovide insight into ways for improving transplantation success ofhematopoietic and progenitor stem cells. The present invention satisfiesthese, and other, needs.

SUMMARY OF THE INVENTION

Methods are provided to manipulate phagocytosis of hematopoietic cells,including circulating hematopoietic cells, e.g. bone marrow cells. Insome embodiments of the invention the circulating cells arehematopoietic stem cells, or hematopoietic progenitor cells,particularly in a transplantation context, where protection fromphagocytosis is desirable. In other embodiments the circulating cellsare leukemia cells, particularly acute leukemia cells such as AML (acutemyeloid leukemia) or ALL (acute lymphocytic leukemia), where increasedphagocytosis is desirable. In certain embodiments of the invention,methods are provided to manipulate macrophage phagocytosis ofcirculating hematopoietic cells. In yet other embodiments of theinvention, methods are provided to manipulate phagocytosis of solidtumors.

In other embodiments, tumor cells, e.g. solid tumor cells, leukemiacells, etc. are targeted for phagocytosis by blocking CD47 on the cellsurface. It is shown that leukemia cells, particularly AML, ALL, etc.cells, evade macrophage surveillance by upregulation of CD47 expression.Administration of agents that mask the CD47 protein, e.g. antibodiesthat bind to CD47 and prevent interaction between CD47 and SIRP□ areadministered to a patient, which increases the clearance of acuteleukemia cells via phagocytosis. In other embodiments, cells of solidtumors, e.g. carcinoma cells, are targeted for phagocytosis by blockingCD47 present on the cell surface. In other aspects, an agent that masksCD47 is combined with monoclonal antibodies directed against one or moreadditional leukemia stem cell (LSC) markers, e.g. CD96, and the like,which compositions can be synergistic in enhancing phagocytosis andelimination of LSC as compared to the use of single agents.

In another embodiment, methods are provided for targeting or depletingleukemia stem cells, the method comprising contacting a population ofcells, e.g. blood from a leukemia patient, with a reagent thatspecifically binds CD47 in order to target or deplete LSC. In certainaspects, the reagent is an antibody conjugated to a cytotoxic agent,e.g. radioactive isotope, chemotherapeutic agent, toxin, etc. In someembodiments, the depletion is performed on an ex vivo population ofcells, e.g. the purging of autologous stem cell products (mobilizedperipheral blood or bone marrow) for use in autologous transplantationfor patients with acute myeloid leukemia. In another embodiment, methodsare provided for targeting cancer cells of a solid tumor in a humansubject by administering an antibody against CD47 to the subject.

In some embodiments of the invention, hematopoietic stem or progenitorcells are protected from phagocytosis in circulation by providing a hostanimal with a CD47 mimetic molecule, which interacts with SIRP□ onphagocytic cells, such as, macrophages, and decreases phagocytosis. TheCD47 mimetic may be soluble CD47; CD47 coated on the surface of thecells to be protected, a CD47 mimetic that binds to SIRP□ at the CD47binding site, and the like. In some embodiments of the invention, CD47is provided as a fusion protein, for example soluble CD47 fused to an Fcfragment, e.g., IgG1 Fc, IgG2 Fc, Ig A Fc etc.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. FACS analysis of human HSC and progenitor CD47 expression fromMyelodysplastic syndrome (MDS, blue), Chronic Myelogenous Leukemia,Accelerated Phase (CIVIL AP, green) and normal bone marrow (red).

FIG. 2. ET vs. PV. FACS analysis of CD47 expression by humanmyeloproliferative disorders such as essential thrombocythemia (ET,blue) and polycythemia vera (PV, green) HSC, progenitor and lineagepositive cells compared with human normal bone marrow (red).

FIG. 3A. Progenitor Profiles of Normal Bone Marrow (left),post-polycythemic myelofibrosis with myeloid metaplasia (PPMM) and CIVILBlast Crisis. FIG. 3B. FACS analysis of human normal bone marrow (red)versus UMPD (green) versus PV (blue=ML) versus atypical CML (orange),HSC, progenitor and lineage positive cell CD47 expression.

FIG. 4. Increased CD47 Expression by CMML Progenitors (blue) comparedwith normal bone marrow (red) with disease progression.

FIG. 5A-5B. (FIG. 5A) Progenitor Profiles of Normal bone marrow (left)versus AML (right). (FIG. 5B) FACS analysis of human normal bone marrow(red) versus AML (blue) HSC, progenitor and lineage positive cell(blast) CD47 expression.

FIG. 6A-6B. CD47 is More Highly Expressed on AML LSC Compared to TheirNormal Counterparts. FIG. 6A. Relative CD47 expression on normal bonemarrow HSC (Lin−CD34+CD38−CD90+) and MPP (Lin−CD34+CD38−CD90-CD45RA−),as well as LSC (Lin−CD34+CD38−CD90−) and bulk leukemia cells from humanAML samples was determined by flow cytometry. Mean fluorescenceintensity was normalized for cell size and against lineage positivecells to account for analysis on different days. The same sample ofnormal bone marrow (red, n=3) or AML (blue, n=13) is indicated by thesame symbol in the different populations. The differences between themean expression of HSC with LSC (p=0.003), HSC with bulk leukemia(p=0.001), MPP with LSC (p=0.004), and MPP with bulk leukemia (p=0.002)were statistically significant using a 2-sided Student's t-test. Thedifference between the mean expression of AML LSC compared to bulk AMLwas not statistically significant with p=0.50 using a paired 2-sidedStudent's t-test. FIG. 6B. Clinical and molecular characteristics ofprimary human AML samples manipulated in vitro and/or in vivo.

FIG. 7A-7C. Anti-CD47 Antibody Stimulates In Vitro MacrophagePhagocytosis of Primary Human AML LSC. AML LSC were purified by FACSfrom two primary human AML samples, labeled with the fluorescent dye CFSE, and incubated with mouse bone marrow-derived macrophages either inthe presence of an isotype control (FIG. 7A) or anti-CD47 antibody (FIG.7B). These cells were assessed by immunofluorescence microscopy for thepresence of fluorescently labeled LSC within the macrophages. (FIG. 7C)The phagocytic index was determined for each condition by calculatingthe number of ingested cells per 100 macrophages.

FIG. 8A-8C. Monoclonal Antibodies Directed Against Human CD47Preferentially Enable Phagocytosis of Human AML LSC by Human and MouseMacrophages. FIG. 8A,8B. CFSE-labeled AML LSC were incubated with humanperipheral blood-derived macrophages (FIG. 8A) or mouse bonemarrow-derived macrophages (FIG. 8B) in the presence of IgG1 isotypecontrol, anti-CD45 IgG1, or anti-CD47 (B6H12.2) IgG1 antibody. Thesecells were assessed by immunofluorescence microscopy for the presence offluorescently labeled LSC within the macrophages (indicated by arrows).FIG. 8C. CFSE-labeled AML LSC or normal bone marrow CD34+ cells wereincubated with human (left) or mouse (right) macrophages in the presenceof the indicated antibodies and then assessed for phagocytosis byimmunofluorescence microscopy. The phagocytic index was determined foreach condition by calculating the number of ingested cells per 100macrophages. For AML LSC, the differences between isotype or anti-CD45antibody with blocking anti-CD47 antibody treatment (B6H12.2 andBRIC126) were statistically significant with p<0.001 for all pairwisecomparisons with human and mouse macrophages. For human macrophages, thedifferences between AML LSC and normal CD34⁺ cells were statisticallysignificant for B6H12.2 (p<0.001) and BRIC126 (p=0.002).

FIG. 9A-9B. Anti-CD47 Antibody stimulates in vitro macrophagephagocytosis of primary human AML LSC. AML LSC were purified by FACSfrom four primary human AML samples, labeled with the fluorescent dyeCFSE, and incubated with human peripheral blood macrophages either inthe presence of an isotype control, isotype matched anti-CD45, oranti-CD47 antibody. (FIG. 9A) These cells were assessed byimmunofluorescence microscopy for the presence of fluorescently-labeledLSC within the macrophages. The phagocytic index was determined for eachcondition by calculating the number of ingested cells per 100macrophages. (FIG. 9B) The macrophages were harvested, stained with afluorescently labeled anti-human macrophage antibody, and analyzed byflow cytometry. hMac+CFSE+ double positive events identify macrophagesthat have phagocytosed CFSE-labeled LSC. Each sample is represented by adifferent color.

FIG. 10A-10B: A Monoclonal Antibody Directed Against Human CD47 InhibitsAML LSC Engraftment In Vivo. Three primary human AML samples wereincubated with IgG1 isotype control, anti-CD45 IgG1, or anti-CD47 IgG1antibody (B6H12.2) prior to transplantation into newborn NOG mice. Aportion of the cells was analyzed for coating by staining with asecondary anti-mouse IgG antibody and analyzed by flow cytometry (FIG.10A). 13 weeks later, mice were sacrificed and the bone marrow wasanalyzed for the percentage of human CD45+CD33+ myeloid leukemia cellsby flow cytometry (FIG. 10B). The difference in mean engraftment betweenanti-CD47-coated cells and both isotype (p<0.001) and anti-CD45(p=0.003) coated cells was statistically significant.

FIG. 11A-11F. CD47 is upregulated in murine acute myeloid leukemia.Typical stem and progenitor plots are shown for leukemichMRP8bcrabl×hMRP8bcl2 cells compared to control non-leukemic animals.Lin− c-Kit+ Sca-1+ gated cells from control bone marrow (FIG. 11A) andleukemic spleen (FIG. 11B) and Lin− c-Kit+ Sca-1− gated cells fromcontrol bone marrow (FIG. 11C) and leukemic spleen (FIG. 11D)demonstrate perturberances in normal hematopoiesis in leukemic mice.Frequency is shown as a percentage of entire marrow or spleenmononuclear fraction. (FIG. 11E) Quantitative RT-PCR shows that CD47 isupregulated in leukemic BM cells. Data are shown from 3 sets of micetransplanted with either leukemic or control hRMP8bcrabl×hMRP8bcl2 BMcells and then sacrificed 2-6 weeks later. Results were normalized tobeta-actin and 18S rRNA expression. Fold change relative to controltransplanted whole Bcl-2+ BM cells was determined. Error bars represent1 s.d. (FIG. 11F) Histograms show expression of CD47 on gatedpopulations for leukemic (gray) and control (black) mice.

FIG. 12A-12C. GMP expansion and CD47 upregulation in human myeloidleukemia. FIG. 12A Representative FACS plots of myeloid progenitors(CD34+CD38+Lin−) including common myeloid progenitors (CMP),megakaryocyte-erythroid progenitors (MEP) and granulocyte-macrophageprogenitors (GMP) in normal bone marrow (BM) versus aCML, BC CIVIL andAML. FIG. 12B Comparative FACS histograms of CD47 expression by normal(red; n=6) and acute myelogenous leukemic (AML, blue; n=6) hematopoieticstem cells (HSC; CD34+CD38− CD90+Lin−) and progenitors (CD34+CD38+Lin−).FIG. 12C Comparative FACS histograms of CD47 expression by normal (red)and chronic myelogenous leukemia hematopoietic stem cells (HSC;CD34+CD38−CD90+Lin) and committed progenitors (CD34+CD38+Lin−). Upperpanel: Normal (n=7) versus chronic phase CIVIL (n=4) HSC, progenitorsand lineage positive cells. Middle panel: Normal (n=7) versusaccelerated phase CIVIL (n=7) HSC, progenitors and lineage positivecells. Lower panel: Normal (n=7) versus blast crisis CML (n=4) HSC,progenitors and lineage positive cells.

FIG. 13A-13G. Over-expression of murine CD47 increases tumorigenicity ofMOLM-13 cells. FIG. 13A MOLM-13 cells were transduced with eithercontrol virus or virus expressing murine CD47 cDNA form 2. The resultingcell lines, termed Tet or Tet-CD47, were transplanted competitively intoRAG/common gamma chain deficient mice with untransduced MOLM-13 cells(5×10⁵ Tet (n=6) or Tet-47 (n=8) cells with 5×10⁵ MOLM-13). Mice wereanalyzed for GFP and human CD45 chimerism when moribund. FIG. 13BMOLM-13 chimerism in hematopoietic tissues was determined by human CD45chimerism and measurement of tumor lesion size. FIG. 13C Survival ofmice competitively transplanted with MOLM-13 plus Tet or Tet-CD47MOLM-13 cells was plotted. Control mice died of large tumor burden atthe site of injection but had no engraftment in hematopoietic tissues.FIG. 13D Hematoxylin and eosin sections of Tet-CD47 MOLM-13 transplantedliver (200×). Periportal (arrow) and sinusoidal (arrowhead) tumorinfiltration is evident. FIG. 13E 1×10⁶ Tet (n=5) or Tet-CD47 MOLM-13(n=4) cells were injected into the right femur of RAG2−/−, Gc−/− miceand the tissues were analyzed 50-75 days later and chimerism of MOLM-13cells in bone marrow was determined. FIG. 13F Survival curve of micetransplanted intrafemorally with Tet or Tet-CD47 MOLM-13 cells. FIG. 13GExamples of liver tumor formation and hepatomegaly in Tet-CD47 MOLM-13transplanted mice versus control transplanted mice. GFP fluorescencedemonstrates tumor nodule formation as well diffuse infiltration.

FIG. 14A-14D. CD47 over-expression prevents phagocytosis of liveunopsonized MOLM-13 cells. FIG. 14A Tet or Tet-CD47 MOLM-13 cells wereincubated with bone marrow derived macrophages (BMDM) for 2, 4, or 6hours and phagocytic index was determined. Error bars represent 1 s.d.(n=6 for each time point). FIG. 14B FACS analysis of BMDMs incubatedwith either Tet or Tet-CD47 cells. FIG. 14C Photomicrographs of BMDMsincubated with Tet or Tet-CD47 MOLM-13 cells at 2 and 24 hours (400×).FIG. 14D Tet or Tet-CD47 MOLM-13 cells were transplanted into RAG2−/−,Gc−/− mice and marrow, spleen, and liver macrophages were analyzed 2hours later. GFP+ fraction of macrophages are gated. Results arerepresentative of 3 experiments.

FIG. 15A-15I. Higher expression of CD47 on MOLM-13 cells correlates withtumorigenic potential and evasion of phagocytosis. FIG. 15A Tet-CD47MOLM-13 cells were divided into high and low expressing clones asdescribed. Histograms show CD47 expression in MOLM-13 high (black),MOLM-13 low (gray), and mouse bone marrow (shaded) cells. Value obtainedfor MFI/FSC² (×10⁹) are shown. FIG. 15B Mice transplanted with CD47hiMOLM-13 cells were given doxycycline for 2 weeks. The histograms showlevel of CD47 expression in untreated (shaded) and treated (shaded)mice, with the values of MFI/FSC² (×10⁹) indicated. FIG. 15C Survival ofRAG2−/−, Gc−/− mice transplanted with 1×10⁶ CD47^(hi), CD47^(lo) MOLM-13cells, or CD47^(hi) MOLM-13 cells with doxycycline administration after2 weeks post-transplant. FIG. 15D Liver and spleen size of mice atnecropsy or 75 days after transplant with 1×10⁶ CD47^(lo), CD47^(lo)MOLM-13 cells, or CD47^(lo) MOLM-13 cells with doxycyclineadministration after 2 weeks post-transplant. FIG. 15E Bone marrow andspleen chimerism of human cells in mice at necropsy or 75 days aftertransplant with 1×10⁶ CD47^(lo), CD47^(lo) MOLM-13 cells, or CD47^(loi)MOLM-13 cells with doxycycline administration after 2 weekspost-transplant. FIG. 15F Murine CD47 expression on CD47^(lo) MOLM-13cells engrafting in bone marrow (open) compared with original cell line(shaded). The values of MFI/FSC² (×10⁹) are indicated. FIG. 15G 2.5×10⁵CD47^(hi) or CD47^(lo) MOLM-13 cells were incubated with 5×10⁴ BMDMs for2 hours. Phagocytic index is shown. FIG. 15H 2.5×10⁵ CD47^(hi) RFP andCD47^(lo) MOLM-13 GFP cells were incubated with 5×10⁴ BMDMs for 2 hours.Phagocytic index is shown for three separate samples for CD47^(lo) RFP(red) and CD47^(lo) MOLM-13 GFP (green) cells. FIG. 15I 2.5×10⁵CD47^(hi) RFP and CD47^(lo) MOLM-13 GFP cells were incubated with 5×10⁴BMDMs for 24 hours. Photomicrographs show brightfield (top left), RFP(top right), GFP (bottom left), and merged (bottom right) images.

FIG. 16A-16C. FIG. 16A FACS analysis of CD47 expression of non-leukemicFas lpr/lpr hMRP8bcl-2 (blue) and leukemic Fas lpr/lpr hMRP8bcl-2(green) bone marrow hematopoietic stem cells (c-kit+Sca+Lin−), myeloidprogenitors (c-kit+Sca−Lin−) or blasts (c-kit lo Sca−Lin−). FIG. 16BMouse bone marrow was transduced with retrovirus containing p210 bcr/ablas previously described²⁴. Mice were sacrificed when moribund and thespleens were analyzed. Expression of CD47 in c-Kit+Mac-1+ cells in thespleens of two leukemic mice (unshaded histograms) and bone marrow froma wild-type mouse (shaded histogram) are shown. FIG. 16C Histograms showexpression of CD47 on gated populations for leukemichMRP8bcrabl×hMRP8bcl2 mice (red), hMRP8bcl2 non-leukemic (blue) andwild-type (green) mice. CD47 was stained using FITC conjugatedanti-mouse CD47 (Pharmingen).

FIG. 17A-17D. FIG. 17A Expression of human CD47 (black histograms) onhuman leukemia cell lines and cord blood HSCs is shown. Isotype controlstaining is shown in gray. FIG. 17B CD47 MFI over background wasnormalized to cell size by dividing by FSC². The value obtained for eachcell type is shown above the bar. FIG. 17C HL-60 cells engraft mousebone marrow. 5×10⁵ cells were injected intravenously into RAG2−/−, Gc−/−animals and mice were analyzed 4 weeks later. FIG. 17D Cells werestained with CFSE and co-cultured with BMDM. Phagocytic events werecounted after 2 h. For irradiation, Jurkat cells were given a dose of 2Gray and incubated for 16 h prior to the phagocytosis assay.

FIG. 18A-18G. FIG. 18A Analysis of stem and progenitor cells from bonemarrow of IAP+/+, IAP+/−, and IAP−/− mice. Stem cells (left) are gatedon lineage− c-Kit+ Sca-1+ cells. Myeloid progenitors (right) are gatedon lineage− c-Kit+ Sca-1+ cells. Frequency in whole bone marrow is shownadjacent to each gated population. FIG. 18B Colony output on day 7 ofindividually sorted LT-HSC. G—granulocyte, M—macrophage, GM—granulocyteand macrophage, GEMM—granulocyte, macrophage, erythroid, andmegakaryocyte, Meg—megakaryocyte. FIG. 18C Survival curve of recipientmice given a radiation dose of 9.5 Gray and transplanted with the cellsshown. Radiation control mice all died within 12-15 days. n=5 for eachgroup. FIG. 18D Examples of CD45.1/CD45.2 chimerism plots at 4 weekspost-transplant. CD45.1 mice were transplanted with 50 LT-HSC (CD45.2)and 2×10⁵ CD45.1 helper marrow. Cells are gated on B220− CD3− Mac-1+side scatter mid/hi cells. IAP−/− cells fail to engraft. FIG. 18ESummary of chimerism analysis of mice transplanted with either 50 or 500IAP+/+ or IAP−/− cells. FIG. 18F IAP+/+ or IAP−/− c-Kit enriched cellswere incubated with wild-type BMDM. Results indicate mean phagocyticindex calculated from three separate samples. Error bars represent 1s.d. FIG. 18G Photomicrographs of phagocytosis assays taken after 2hours. Genotype of the -Kit enriched cells is shown.

FIG. 19A-19E. FIG. 19A Mice were mobilized with Cy/G and bone marrow wasanalyzed on day 2. Expression level of CD47 on c-Kit+ cells is shown.FIG. 19B Myeloid progenitor and stem cell gates are shown for day 2mobilized bone marrow. Histograms on left show level of CD47 expressionin marrow LT-HSC and GMP for steady-state (shaded histogram), day 2mobilized (black line), and day 5 mobilized (gray line). FIG. 19CRelative MFI of CD47 for GMP on days 0-5 of Cy/G mobilization. Resultswere normalized so that steady state GMP were equal to 100. FIG. 19DMyeloid progenitor and stem cell gates are shown for day 2 bone marrowpost-LPS treatment. Histograms show level of CD47 expression on day 2post-LPS (black line), day 5 post-LPS (dark gray shaded histogram),steady state (light gray shaded histogram), and IAP−/− (black shadedhistogram) LT-HSC and GMP. FIG. 19E Evaluation of KLS cells in thehematopoietic organs of IAP+/+ and IAP−/− mice mobilized on days 2through 5. Two mice are analyzed per genotype per day.

FIG. 20A-20B. FIG. 20A CD47 expression level of IAP+/+, IAP+/−, andIAP−/− LT-HSC. The numbers shown are the MFI for each group. FIG. 20BDonor chimerism analysis for transplants of IAP+/+(top) or IAP+/−(bottom) mice. Mice were bled at 2, 8, and 40 weeks post transplant.2×10⁶ donor cells were transplanted into sub-lethally irradiatedcongenic recipients.

FIG. 21A-21D: Identification and Separation of Normal and LeukemicProgenitors From the Same Patient Based On Differential CD47 Expression.FIG. 21A. CD47 expression on the Lin−CD34+CD38− LSC-enriched fraction ofspecimen SU008 was determined by flow cytometry. CD47hi- andCD47lo-expressing cells were identified and purified using FACS. Theleft panels are gated on lineage negative cells, while the right panelsare gated on Lin−CD34+CD38− cells. FIG. 21B. Lin−CD34+CD38−CD47lo andLin−CD34+CD38−CD47hi cells were plated into complete methylcellulose,capable of supporting the growth of all myeloid colonies. 14 days later,myeloid colony formation was determined by morphologic assessment.Representative CFU-G/M (left) and BFU-E (right) are presented. FIG. 21C.Lin−CD34+CD38−CD47lo cells were transplanted into 2 newborn NOG mice. 12weeks later, the mice were sacrificed and the bone marrow was analyzedfor the presence of human CD45+CD33+ myeloid cells and human CD45+CD19+lymphoid cells by flow cytometry. FIG. 21D. Normal bone marrow HSC, bulkSU008 leukemia cells, Lin−CD34+CD38−CD47hi cells, Lin−CD34+CD38−CD47locells, or human CD45+ cells purified from the bone marrow of miceengrafted with Lin−CD34+CD38− CD47lo cells were assessed for thepresence of the FLT3-ITD mutation by PCR. The wild type FLT3 and theFLT3-ITD products are indicated.

FIG. 22A-22D: Increased CD47 Expression in Human AML is Associated withPoor Clinical Outcomes. Event-free (FIG. 22A,22C) and overall (FIG.22B,22D) survival of 132 AML patients with normal cytogenetics (FIG.22A,22B) and the subset of 74 patients without the FLT3-ITD mutation(FIG. 22C,22D). Patients were stratified into low CD47 and high CD47expression groups based on an optimal threshold (28% high, 72% low)determined by microarray analysis from an independent training data set.The significance measures are based on log-likelihood estimates of thep-value, when treating the model with CD47 expression as a binaryclassification.

FIG. 23A-23E: A Monoclonal Antibody Directed Against Human CD47Eliminates AML In Vivo. Newborn NOG mice were transplanted with AML LSC,and 8-12 weeks later, peripheral blood (FIG. 23A,23B) and bone marrow(FIG. 23C-23E) were analyzed for baseline engraftment prior to treatmentwith anti-CD47 (B6H12.2) or control IgG antibody (Day 0). Mice weretreated with daily 100 microgram intraperitoneal injections for 14 days,at the end of which, they were sacrificed and peripheral blood and bonemarrow were analyzed for the percentage of human CD45+CD33+ leukemia.FIG. 23A. Pre- and post-treatment human leukemic chimerism in theperipheral blood from representative anti-CD47 antibody and controlIgG-treated mice as determined by flow cytometry. FIG. 23B. Summary ofhuman leukemic chimerism in the peripheral blood assessed on multipledays during the course of treatment demonstrated elimination of leukemiain anti-CD47 antibody treated mice compared to control IgG treatment(p=0.007). FIG. 23C. Pre- and post-treatment human leukemic chimerism inthe bone marrow from representative anti-CD47 antibody or controlIgG-treated mice as determined by flow cytometry. FIG. 23D. Summary ofhuman leukemic chimerism in the bone marrow on day 14 relative to day 0demonstrated a dramatic reduction in leukemic burden in anti-CD47antibody treated mice compared to control IgG treatment (p<0.001). FIG.23E. H&E sections of representative mouse bone marrow cavities from miceengrafted with SU004 post-treatment with either control IgG (panels 1,2)or anti-CD47 antibody (panels 4,5). IgG-treated marrows were packed withmonomorphic leukemic blasts, while anti-CD47-treated marrows werehypocellular, demonstrating elimination of the human leukemia. In someanti-CD47 antibody-treated mice that contained residual leukemia,macrophages were detected containing phagocytosed pyknotic cells,capturing the elimination of human leukemia (panels 3,6 arrows).

FIG. 24A-24B. Increased CD47 expression predicts worse overall survivalin DLBCL and ovarian cancer. (FIG. 24A) A cohort of 230 patients withdiffuse large B-cell lymphoma (p=0.01). (FIG. 24B) A cohort of 133patients with advanced stage (III/IV) ovarian carcinoma (p=0.04).

FIG. 25 Anti-CD47 antibody enables the phagocytosis of solid tumor stemcells in vitro. The indicated cells were incubated with humanmacrophages in the presence of IgG1 isotype, anti-HLA, or anti-CD47antibodies and the phagocytic index was determined by immunofluorescencemicroscopy. Statistics: Bladder cancer cells IgG1 isotype compared toanti-HLA (p=0.93) and anti-CD47 (p=0.01); normal bladder urothelium IgG1isotype compared to anti-HLA (p=0.50) and anti-CD47 (p=0.13); ovariancancer cells IgG1 isotype compared to anti-HLA (p=0.11) and anti-CD47(p<0.001). Each individual data point represents a distinct tumor ornormal tissue sample.

FIG. 26A-26D: CD47 expression is an independent prognostic predictor inmixed and high-risk ALL. (FIG. 26A) Pediatric ALL patients (n=360) withmixed risk and treatment were stratified into CD47 high- andlow-expressing groups based on an optimal cut point. Disease-freesurvival (DFS) was determined by Kaplan-Meier analysis. CD47high-expressing patients had a worse DFS compared to CD47 low-expressingpatients when CD47 expression was considered as a continuous variable.(FIG. 26B) Pediatric ALL patients (n=207) with high-risk (as defined byage>10 years, presenting WBC count>50,000/μl, hypodiploidy, and BCR-ABLpositive disease) and uniform treatment were stratified into CD47 high-and low-expressing groups using a similar approach as in FIG. 26A. CD47high-expressing patients had a worse overall survival compared to CD47low-expressing patients (p=0.0009). (FIG. 26C) Multivariate analysis ofprognostic covariates was performed from patients analyzed for CD47expression in (FIG. 26B). When incorporated into this multivariateanalysis, CD47 expression still remained prognostic (p=0.035). (FIG.26D) ALL patients from were stratified into groups either achieving acomplete remission (CR) or not achieving a CR (no CR). CD47 expressionwas higher in patients failing to receive a CR compared to those who did(p=0.0056).

FIG. 27A-27C: Ex vivo coating of ALL cells with an anti-CD47 antibodyinhibits leukemic engraftment. (FIG. 27A) ALL cells were incubated withthe indicated antibodies in vitro, and positive cell coating wasdetected by staining a portion of the cells with a fluorescently-labeledsecondary antibody. A flow cytometry plot of a representative ALL sampleis shown. (FIG. 27B-27C) Pre-coated ALL cells were then transplantedinto NSG mice, and human ALL chimerism was assessed 6-10 weeks later inthe peripheral blood (FIG. 27B) or bone marrow (FIG. 27C). Ex vivocoating of ALL cells (ALL4 and ALL8) with anti-CD47 antibody inhibitedengraftment in the peripheral blood compared to IgG1 isotype control(p=0.02). Ex vivo coating of ALL cells with anti-CD47 antibody inhibitedbone marrow engraftment compared to IgG1 isotype control (p=0.02), whileno difference in engraftment levels were detected between anti-CD45antibody and IgG1 isotype control (p=0.67, considering both B and T-ALLsamples). Each symbol represents a different primary ALL sample, witheach point representing a different mouse. p-values were calculatedusing the Fisher's exact test. Red diamond=ALL4, blue diamond=ALL4.

FIG. 28A-28D: Anti-CD47 antibody eliminates ALL engraftment in theperipheral blood and bone marrow. (FIG. 28A) NSG mice engrafted withprimary B and T-ALL patient samples were treated for 14 days with dailyintraperitoneal injections of 100 μg IgG control or anti-CD47 antibody.Peripheral blood human ALL chimerism (huCD45+CD19/CD3+) pre- andpost-treatment were measured by flow cytometry. Peripheral bloodchimerism is shown from representative treatment mice. (FIG. 28B)Anti-CD47 antibody treatment reduced the level of circulating leukemiacompared to IgG control (p=0.0002). (FIG. 28C) Anti-CD47 antibodytreatment also reduced ALL engraftment in the bone marrow compared toIgG control (p=0.0004). Each symbol represents a different patientsample, with each data point representing a different mouse. (FIG. 28D)(Top) Hematoxylin and eosin bone marrow sections from representativemice engrafted with B-ALL post-treatment. IgG-treated marrows wereprimarily packed with monomorphic leukemic blasts, while anti-CD47antibody-treated marrows demonstrated areas of normal mousehematopoiesis. (Bottom) Leukemic infiltration was confirmed byimmunohistochemical analysis of human CD45 demonstrating robust humanleukemia infiltration in IgG-treated bone marrow compared to anti-CD47antibody-treated marrow.

FIG. 29A-29D: Anti-CD47 antibody eliminates ALL engraftment in thespleen and liver. (FIG. 29A) NSG mice engrafted with primary B-ALL cellsfrom sample ALL8, ALL21, or ALL22 were treated for 14 days with dailyinjections of IgG control or anti-CD47 antibody. Spleens were thenharvested, with representative spleens from IgG control or anti-CD47antibody treatment shown. (FIG. 29B) Spleen weights were determined frommice treated with anti-CD47 antibody demonstrating a reduction in spleensize compared to control IgG-treated mice (p=0.04) to sizes similar tothat of normal spleens (p=0.09). Control IgG-treated mice demonstratesplenomegaly compared to normal mice (p=0.0002, student t-test). (FIG.29C-29D) Levels of ALL engraftment were determined at the end ofantibody treatment in the spleen (FIG. 29C) and liver (FIG. 29D).Compared to IgG control, treatment with anti-CD47 antibody eliminatedALL disease in the spleen (p<0.0001) and liver (p<0.0001, studentt-test).

FIG. 30A-30D: Antibodies targeted to human CD47 enable the phagocytosisof human cancer cells. FIG. 30A: CFSE-labeled human patient bladdercancer cells were incubated with human macrophages in the presence ofthe indicated antibodies and assessed for the presence of tumor cellswithin macrophages. FIG. 30B-30D: Phagocytosis of patient bladder cancercells (FIG. 30B), ovarian cancer cells (FIG. 30C), or colon cancer stemcells (FIG. 30D) resulting from indicated antibody treatment wasquantified. Each dot color represents a different primary tumor sample.Open (non-colored) symbols represent normal tissue controls. Thephagocytic index was determined as the number of CFSE labeled tumorcells present within 100 macrophages.

FIG. 31A-31F: Antibodies targeted to CD47 inhibit the growth of patienttumors. FIG. 31A-31D: Tumor cells from ovarian (FIG. 31A), pancreatic(FIG. 31B), breast (FIG. 31C), or colon (FIG. 31D) tumors were engraftedinto immunodeficient mice. These mice were then treated with control IgGor anti-CD47 antibodies and tumor growth was assessed directly or bybioluminescence. In all cases, anti-CD47 antibody treatmentsubstantially inhibited tumor growth. FIG. 31E-31F: Tumor cells frompatient bladder were injected subcutaneously into immunodeficient miceand treated with the indicated antibodies. Anti-CD47 antibody treatmentsignificantly inhibited metastasis to the lymph nodes (FIG. 31E) andformation of micrometastases in the lungs (FIG. 31F). The total numberof metastases detected in each treatment group is indicated.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Methods are provided to manipulate the phagocytosis of cells, includingcirculating hematopoietic cells. In some embodiments of the invention,leukemia cells, e.g. AML, B-ALL, T-ALL, etc. are targeted forphagocytosis by blocking CD47 on the cell surface. In other embodiments,cells of solid tumors are targeted for phagocytosis by blocking CD47 onthe cell surface. In another embodiment, methods are provided fortargeting or depleting leukemia stem cells, e.g. AML stem cells, ALLstem cells, etc., the method comprising contacting reagent blood cellswith an antibody that specifically binds CD47 in order to target ordeplete LSC.□ In another embodiment, methods are provided for targetingcancer cells of a tumor in a human subject by administering an antibodyspecific for CD47 to the subject.

In other embodiments, hematopoietic stem or progenitor cells areprotected from phagocytosis in circulation by providing a host animalwith a CD47 mimetic molecule, which interacts with SIRP□ on phagocyticcells, such as, macrophages, and decreases phagocytosis.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Methods recited herein may be carried out in any order of the recitedevents which is logically possible, as well as the recited order ofevents.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DEFINITIONS

CD47 polypeptides. The three transcript variants of human CD 47 (variant1, NM 001777 (SEQ ID NO: 2); variant 2, NM 198793 (SEQ ID NO: 3); andvariant 3, NM 001025079 (SEQ ID NO: 4)) encode three isoforms of CD47polypeptide. CD47 isoform 1 (NP 001768; SEQ ID NO: 5), the longest ofthe three isoforms, is 323 amino acids long. CD47 isoform 2 (NP 942088;SEQ ID NO: 6) is 305 amino acid long. CD47 isoform 3 (SEQ ID NO: 7) is312 amino acids long. The three isoforms are identical in sequence inthe first 303 amino acids. Amino acids 1-8 comprise the signal sequence,amino acids 9-142 comprise the CD47 immunoglobulin like domain, which isthe soluble fragment, and amino acids 143-300 is the transmembranedomain.

“CD47 mimetics” include molecules that function similarly to CD47 bybinding and activating SIRP□ receptor. Molecules useful as CD47 mimeticsinclude derivatives, variants, and biologically active fragments ofnaturally occurring CD47. A “variant” polypeptide means a biologicallyactive polypeptide as defined below having less than 100% sequenceidentity with a native sequence polypeptide. Such variants includepolypeptides wherein one or more amino acid residues are added at the N-or C-terminus of, or within, the native sequence; from about one toforty amino acid residues are deleted, and optionally substituted by oneor more amino acid residues; and derivatives of the above polypeptides,wherein an amino acid residue has been covalently modified so that theresulting product has a non-naturally occurring amino acid. Ordinarily,a biologically active variant will have an amino acid sequence having atleast about 90% amino acid sequence identity with a native sequencepolypeptide, preferably at least about 95%, more preferably at leastabout 99%. The variant polypeptides can be naturally or non-naturallyglycosylated, i.e., the polypeptide has a glycosylation pattern thatdiffers from the glycosylation pattern found in the correspondingnaturally occurring protein. The variant polypeptides can havepost-translational modifications not found on the natural CD47 protein.

Fragments of the soluble CD47, particularly biologically activefragments and/or fragments corresponding to functional domains, are ofinterest. Fragments of interest will typically be at least about 10 aato at least about 15 aa in length, usually at least about 50 aa inlength, but will usually not exceed about 142 aa in length, where thefragment will have a stretch of amino acids that is identical to CD47. Afragment “at least 20 aa in length,” for example, is intended to include20 or more contiguous amino acids from, for example, the polypeptideencoded by a cDNA for CD47. In this context “about” includes theparticularly recited value or a value larger or smaller by several (5,4, 3, 2, or 1) amino acids. The protein variants described herein areencoded by polynucleotides that are within the scope of the invention.The genetic code can be used to select the appropriate codons toconstruct the corresponding variants. The polynucleotides may be used toproduce polypeptides, and these polypeptides may be used to produceantibodies by known methods.

A “fusion” polypeptide is a polypeptide comprising a polypeptide orportion (e.g., one or more domains) thereof fused or bonded toheterologous polypeptide. A fusion soluble CD47 protein, for example,will share at least one biological property in common with a nativesequence soluble CD47 polypeptide. Examples of fusion polypeptidesinclude immunoadhesins, as described above, which combine a portion ofthe CD47 polypeptide with an immunoglobulin sequence, and epitope taggedpolypeptides, which comprise a soluble CD47 polypeptide or portionthereof fused to a “tag polypeptide”. The tag polypeptide has enoughresidues to provide an epitope against which an antibody can be made,yet is short enough such that it does not interfere with biologicalactivity of the CD47 polypeptide. Suitable tag polypeptides generallyhave at least six amino acid residues and usually between about 6-60amino acid residues.

A “functional derivative” of a native sequence polypeptide is a compoundhaving a qualitative biological property in common with a nativesequence polypeptide. “Functional derivatives” include, but are notlimited to, fragments of a native sequence and derivatives of a nativesequence polypeptide and its fragments, provided that they have abiological activity in common with a corresponding native sequencepolypeptide. The term “derivative” encompasses both amino acid sequencevariants of polypeptide and covalent modifications thereof. Derivativesand fusion of soluble CD47 find use as CD47 mimetic molecules.

The first 142 amino acids of CD47 polypeptide comprise the extracellularregion of CD47 (SEQ ID NO: 1). The three isoforms have identical aminoacid sequence in the extracellular region, and thus any of the isoformsare can be used to generate soluble CD47. “Soluble CD47” is a CD47protein that lacks the transmembrane domain. Soluble CD47 is secretedout of the cell expressing it instead of being localized at the cellsurface. Soluble CD47 may be fused to another polypeptide to provide foradded functionality, e.g. to increase the in vivo stability. Generallysuch fusion partners are a stable plasma protein that is capable ofextending the in vivo plasma half-life of soluble CD47 protein whenpresent as a fusion, in particular wherein such a stable plasma proteinis an immunoglobulin constant domain. In most cases where the stableplasma protein is normally found in a multimeric form, e.g.,immunoglobulins or lipoproteins, in which the same or differentpolypeptide chains are normally disulfide and/or noncovalently bound toform an assembled multichain polypeptide. Soluble CD47 fused to human IgG1 has been described (Motegi S. et al. EMBO J. 22(11): 2634-2644).

Stable plasma proteins are proteins typically having about from 30 to2,000 residues, which exhibit in their native environment an extendedhalf-life in the circulation, i.e. greater than about 20 hours. Examplesof suitable stable plasma proteins are immunoglobulins, albumin,lipoproteins, apolipoproteins and transferrin. The extracellular regionof CD47 is typically fused to the plasma protein at the N-terminus ofthe plasma protein or fragment thereof which is capable of conferring anextended half-life upon the soluble CD47. Increases of greater thanabout 100% on the plasma half-life of the soluble CD47 are satisfactory.

Ordinarily, the soluble CD47 is fused C-terminally to the N-terminus ofthe constant region of immunoglobulins in place of the variableregion(s) thereof, however N-terminal fusions may also find use.Typically, such fusions retain at least functionally active hinge, CH2and CH3 domains of the constant region of an immunoglobulin heavy chain,which heavy chains may include IgG1, IgG2a, IgG2b, IgG3, IgG4, IgA, IgM,IgE, and IgD, usually one or a combination of proteins in the IgG class.Fusions are also made to the C-terminus of the Fc portion of a constantdomain, or immediately N-terminal to the CH1 of the heavy chain or thecorresponding region of the light chain. This ordinarily is accomplishedby constructing the appropriate DNA sequence and expressing it inrecombinant cell culture. Alternatively, the polypeptides may besynthesized according to known methods.

The precise site at which the fusion is made is not critical; particularsites may be selected in order to optimize the biological activity,secretion or binding characteristics of CD47. The optimal site will bedetermined by routine experimentation.

In some embodiments the hybrid immunoglobulins are assembled asmonomers, or hetero- or homo-multimers, and particularly as dimers ortetramers. Generally, these assembled immunoglobulins will have knownunit structures. A basic four chain structural unit is the form in whichIgG, IgD, and IgE exist. A four chain unit is repeated in the highermolecular weight immunoglobulins; IgM generally exists as a pentamer ofbasic four-chain units held together by disulfide bonds. IgAimmunoglobulin, and occasionally IgG immunoglobulin, may also exist in amultimeric form in serum. In the case of multimers, each four chain unitmay be the same or different.

Suitable CD47 mimetics and/or fusion proteins may be identified bycompound screening by detecting the ability of an agent to mimic thebiological activity of CD47. One biological activity of CD47 is theactivation of SIRP□ receptor on macrophages. In vitro assays may beconducted as a first screen for efficacy of a candidate agent, andusually an in vivo assay will be performed to confirm the biologicalassay. Desirable agents are effective in temporarily blocking SIRP□□receptor activation. Desirable agents are temporary in nature, e.g. dueto biological degradation.

In vitro assays for CD47 biological activity include, e.g. inhibition ofphagocytosis of porcine cells by human macrophages, binding to SIRP□□receptor, SIRP□□□ tyrosine phosphorylation, etc. An exemplary assay forCD47 biological activity contacts a human macrophage composition in thepresence of a candidate agent. The cells are incubated with thecandidate agent for about 30 minutes and lysed. The cell lysate is mixedwith anti-human SIRP□□□ antibodies to immunoprecipitate SIRP□□□□Precipitated proteins are resolved by SDS PAGE, then transferred tonitrocellulose and probed with antibodies specific for phosphotyrosine.A candidate agent useful as CD47 mimetic increases SIRP□□□ tyrosinephosphorylation by at least 10%, or up to 20%, or 50%, or 70% or 80% orup to about 90% compared to the level of phosphorylation observed in theabsence of candidate agent. Another exemplary assay for CD47 biologicalactivity measures phagocytosis of hematopoietic cells by humanmacrophages. A candidate agent useful as a CD47 mimetic results in thedown regulation of phagocytosis by at least about 10%, at least about20%, at least about 50%, at least about 70%, at least about 80%, or upto about 90% compared to level of phagocytosis observed in absence ofcandidate agent.

Polynucleotide encoding soluble CD47 or soluble CD47-Fc can beintroduced into a suitable expression vector. The expression vector isintroduced into a suitable cell. Expression vectors generally haveconvenient restriction sites located near the promoter sequence toprovide for the insertion of polynucleotide sequences. Transcriptioncassettes may be prepared comprising a transcription initiation region,CD47 gene or fragment thereof, and a transcriptional termination region.The transcription cassettes may be introduced into a variety of vectors,e.g. plasmid; retrovirus, e.g. lentivirus; adenovirus; and the like,where the vectors are able to transiently or stably be maintained in thecells, usually for a period of at least about one day, more usually fora period of at least about several days to several weeks.

The various manipulations may be carried out in vitro or may beperformed in an appropriate host, e.g. E. coli. After each manipulation,the resulting construct may be cloned, the vector isolated, and the DNAscreened or sequenced to ensure the correctness of the construct. Thesequence may be screened by restriction analysis, sequencing, or thelike.

Soluble CD47 can be recovered and purified from recombinant cellcultures by well-known methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, protein G affinity chromatography, for example,hydroxylapatite chromatography and lectin chromatography. Mostpreferably, high performance liquid chromatography (“HPLC”) is employedfor purification.

Soluble CD47 can also be recovered from: products of purified cells,whether directly isolated or cultured; products of chemical syntheticprocedures; and products produced by recombinant techniques from aprokaryotic or eukaryotic host, including, for example, bacterial, yeasthigher plant, insect, and mammalian cells.

A plurality of assays may be run in parallel with differentconcentrations to obtain a differential response to the variousconcentrations. As known in the art, determining the effectiveconcentration of an agent typically uses a range of concentrationsresulting from 1:10, or other log scale, dilutions. The concentrationsmay be further refined with a second series of dilutions, if necessary.Typically, one of these concentrations serves as a negative control,i.e. at zero concentration or below the level of detection of the agentor at or below the concentration of agent that does not give adetectable change in binding.

Compounds of interest for screening include biologically active agentsof numerous chemical classes, primarily organic molecules, althoughincluding in some instances inorganic molecules, organometallicmolecules, immunoglobulins, chimeric CD47 proteins, CD47 relatedproteins, genetic sequences, etc. Also of interest are small organicmolecules, which comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,frequently at least two of the functional chemical groups. The candidateagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules, including peptides, polynucleotides, saccharides, fattyacids, steroids, purines, pyrimidines, derivatives, structural analogsor combinations thereof.

Compounds are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds, including biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial, fungal, plant and animalextracts are available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

By “manipulating phagocytosis” is meant an up-regulation or adown-regulation in phagocytosis by at least about 10%, or up to 20%, or50%, or 70% or 80% or up to about 90% compared to level of phagocytosisobserved in absence of intervention. Thus in the context of decreasingphagocytosis of circulating hematopoietic cells, particularly in atransplantation context, manipulating phagocytosis means adown-regulation in phagocytosis by at least about 10%, or up to 20%, or50%, or 70% or 80% or up to about 90% compared to level of phagocytosisobserved in absence of intervention.

CD47 inhibitors. Agents of interest as CD47 inhibitors include specificbinding members that prevent the binding of CD47 with SIRP□□ receptor.The term “specific binding member” or “binding member” as used hereinrefers to a member of a specific binding pair, i.e. two molecules,usually two different molecules, where one of the molecules (i.e., firstspecific binding member) through chemical or physical means specificallybinds to the other molecule (i.e., second specific binding member). CD47inhibitors useful in the methods of the invention include analogs,derivatives and fragments of the original specific binding member.

In a preferred embodiment, the specific binding member is an antibody.The term “antibody” or “antibody moiety” is intended to include anypolypeptide chain-containing molecular structure with a specific shapethat fits to and recognizes an epitope, where one or more non-covalentbinding interactions stabilize the complex between the molecularstructure and the epitope. Antibodies utilized in the present inventionmay be polyclonal antibodies, although monoclonal antibodies arepreferred because they may be reproduced by cell culture orrecombinantly, and can be modified to reduce their antigenicity.

Polyclonal antibodies can be raised by a standard protocol by injectinga production animal with an antigenic composition. See, e.g., Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,1988. When utilizing an entire protein, or a larger section of theprotein, antibodies may be raised by immunizing the production animalwith the protein and a suitable adjuvant (e.g., Freund's, Freund'scomplete, oil-in-water emulsions, etc.) When a smaller peptide isutilized, it is advantageous to conjugate the peptide with a largermolecule to make an immunostimulatory conjugate. Commonly utilizedconjugate proteins that are commercially available for such use includebovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH). In orderto raise antibodies to particular epitopes, peptides derived from thefull sequence may be utilized. Alternatively, in order to generateantibodies to relatively short peptide portions of the protein target, asuperior immune response may be elicited if the polypeptide is joined toa carrier protein, such as ovalbumin, BSA or KLH. Alternatively, formonoclonal antibodies, hybridomas may be formed by isolating thestimulated immune cells, such as those from the spleen of the inoculatedanimal. These cells are then fused to immortalized cells, such asmyeloma cells or transformed cells, which are capable of replicatingindefinitely in cell culture, thereby producing an immortal,immunoglobulin-secreting cell line. In addition, the antibodies orantigen binding fragments may be produced by genetic engineering.Humanized, chimeric, or xenogeneic human antibodies, which produce lessof an immune response when administered to humans, are preferred for usein the present invention.

In addition to entire immunoglobulins (or their recombinantcounterparts), immunoglobulin fragments comprising the epitope bindingsite (e.g., Fab′, F(ab′)₂, or other fragments) are useful as antibodymoieties in the present invention. Such antibody fragments may begenerated from whole immunoglobulins by ricin, pepsin, papain, or otherprotease cleavage. “Fragment,” or minimal immunoglobulins may bedesigned utilizing recombinant immunoglobulin techniques. For instance“Fv” immunoglobulins for use in the present invention may be produced bylinking a variable light chain region to a variable heavy chain regionvia a peptide linker (e.g., poly-glycine or another sequence which doesnot form an alpha helix or beta sheet motif).

The efficacy of a CD47 inhibitor is assessed by assaying CD47 activity.The above-mentioned assays or modified versions thereof are used. In anexemplary assay, AML SCs are incubated with bone marrow derivedmacrophages, in the presence or absence of the candidate agent. Aninhibitor of the cell surface CD47 will up-regulate phagocytosis by atleast about 10%, or up to 20%, or 50%, or 70% or 80% or up to about 90%compared to the phagocytosis in absence of the candidate agent.Similarly, an in vitro assay for levels of tyrosine phosphorylation ofSIRP□ will show a decrease in phosphorylation by at least about 10%, orup to 20%, or 50%, or 70% or 80% or up to about 90% compared tophosphorylation observed in absence of the candidate agent.

In one embodiment of the invention, the agent, or a pharmaceuticalcomposition comprising the agent, is provided in an amount effective todetectably inhibit the binding of CD47 to SIRP□ receptor present on thesurface of phagocytic cells. The effective amount is determined viaempirical testing routine in the art. The effective amount may varydepending on the number of cells being transplanted, site oftransplantation and factors specific to the transplant recipient.

The terms “phagocytic cells” and “phagocytes” are used interchangeablyherein to refer to a cell that is capable of phagocytosis. There arethree main categories of phagocytes: macrophages, mononuclear cells(histiocytes and monocytes); polymorphonuclear leukocytes (neutrophils)and dendritic cells.

The term “biological sample” encompasses a variety of sample typesobtained from an organism and can be used in a diagnostic or monitoringassay. The term encompasses blood and other liquid samples of biologicalorigin, solid tissue samples, such as a biopsy specimen or tissuecultures or cells derived therefrom and the progeny thereof. The termencompasses samples that have been manipulated in any way after theirprocurement, such as by treatment with reagents, solubilization, orenrichment for certain components. The term encompasses a clinicalsample, and also includes cells in cell culture, cell supernatants, celllysates, serum, plasma, biological fluids, and tissue samples.

Hematopoietic stem cells (HSC), as used herein, refers to a populationof cells having the ability to self-renew, and to give rise to allhematopoietic lineages. Such cell populations have been described indetail in the art. Hematopoietic progenitor cells include the myeloidcommitted progenitors (CMP), the lymphoid committed progenitors (CLP),megakaryocyte progenitors, and multipotent progenitors. The earliestknown lymphoid-restricted cell in adult mouse bone marrow is the commonlymphocyte progenitor (CLP), and the earliest known myeloid-restrictedcell is the common myeloid progenitor (CMP). Importantly, these cellpopulations possess an extremely high level of lineage fidelity in invitro and in vivo developmental assays. A complete description of thesecell subsets may be found in Akashi et al. (2000) Nature 404(6774):193,U.S. Pat. No. 6,465,247; and published application U.S. Ser. No.09/956,279 (common myeloid progenitor); Kondo et al. (1997) Cell91(5):661-7, and International application WO99/10478 (common lymphoidprogenitor); and is reviewed by Kondo et al. (2003) Annu Rev Immunol.21:759-806, each of which is herein specifically incorporated byreference. The composition may be frozen at liquid nitrogen temperaturesand stored for long periods of time, being capable of use on thawing.For such a composition, the cells will usually be stored in a 10% DMSO,50% FCS, 40% RPMI 1640 medium.

Populations of interest for use in the methods of the invention includesubstantially pure compositions, e.g. at least about 50% HSC, at leastabout 75% HSC, at least about 85% HSC, at least about 95% HSC or more;or may be combinations of one or more stem and progenitor cellspopulations, e.g. white cells obtained from apheresis, etc. Wherepurified cell populations are desired, the target population may bepurified in accordance with known techniques. For example, a populationcontaining white blood cells, particularly including blood or bonemarrow samples, are stained with reagents specific for markers presentof hematopoietic stem and progenitor cells, which markers are sufficientto distinguish the major stem and progenitor groups. The reagents, e.g.antibodies, may be detectably labeled, or may be indirectly labeled inthe staining procedure.

Any combination of markers may be used that are sufficient to select forthe stem/progenitor cells of interest. A marker combination of interestmay include CD34 and CD38, which distinguishes hematopoietic stem cells,(CD34⁺, CD38⁻) from progenitor cells, which are CD34⁺, CD38⁺). HSC arelineage marker negative, and positive for expression of CD90.

In the myeloid lineage are three cell populations, termed CMPs, GMPs,and MEPs. These cells are CD34⁺ CD38⁺, they are negative for multiplemature lineage markers including early lymphoid markers such as CD7,CD10, and IL-7R, and they are further distinguished by the markersCD45RA, an isoform of CD45 that can negatively regulate at least someclasses of cytokine receptor signaling, and IL-3R. These characteristicsare CD45RA⁻ IL-3R□^(lo) (CMPs), CD45RA⁺IL-3R□^(lo) (GMPs), and CD45RA⁻IL-3R□^(□) (MEPs). CD45RA⁻ IL-3R□^(lo) cells give rise to GMPs and MEPsand at least one third generate both GM and MegE colonies on asingle-cell level. All three of the myeloid lineage progenitors stainnegatively for the markers Thy-1 (CD90), IL-7R□ (CD127); and with apanel of lineage markers, which lineage markers may include CD2; CD3;CD4; CD7; CD8; CD10; CD11b; CD14; CD19; CD20; CD56; and glycophorin A(GPA) in humans and CD2; CD3; CD4; CD8; CD19; IgM; Ter110; Gr-1 in mice.With the exception of the mouse MEP subset, all of the progenitor cellsare CD34 positive. In the mouse all of the progenitor subsets may befurther characterized as Sca-1 negative, (Ly-6E and Ly-6A), and c-kithigh. In the human, all three of the subsets are CD38⁺.

Common lymphoid progenitors, CLP, express low levels of c-kit (CD117) ontheir cell surface. Antibodies that specifically bind c-kit in humans,mice, rats, etc. are known in the art. Alternatively, the c-kit ligand,steel factor (Slf) may be used to identify cells expressing c-kit. TheCLP cells express high levels of the IL-7 receptor alpha chain (CDw127).Antibodies that bind to human or to mouse CDw127 are known in the art.Alternatively, the cells are identified by binding of the ligand to thereceptor, IL-7. Human CLPs express low levels of CD34. Antibodiesspecific for human CD34 are commercially available and well known in theart. See, for example, Chen et al. (1997) Immunol Rev 157:41-51. HumanCLP cells are also characterized as CD38 positive and CD10 positive. TheCLP subset also has the phenotype of lacking expression of lineagespecific markers, exemplified by B220, CD4, CD8, CD3, Gr-1 and Mac-1.The CLP cells are characterized as lacking expression of Thy-1, a markerthat is characteristic of hematopoietic stem cells. The phenotype of theCLP may be further characterized as Mel-14⁻, CD43^(lo), HSA^(lo), CD45⁺and common cytokine receptor □ chain positive.

Megakaryocyte progenitor cells (MKP) cells are positive for CD34expression, and tetraspanin CD9 antigen. The CD9 antigen is a 227-aminoacid molecule with 4 hydrophobic domains and 1 N-glycosylation site. Theantigen is widely expressed, but is not present on certain progenitorcells in the hematopoietic lineages. The MKP cells express CD41, alsoreferred to as the glycoprotein IIb/IIIa integrin, which is the plateletreceptor for fibrinogen and several other extracellular matrixmolecules, for which antibodies are commercially available, for examplefrom BD Biosciences, Pharmingen, San Diego, Calif., catalog number340929, 555466. The MKP cells are positive for expression of CD117,which recognizes the receptor tyrosine kinase c-Kit. Antibodies arecommercially available, for example from BD Biosciences, Pharmingen, SanDiego, Calif., Cat. No. 340529. MKP cells are also lineage negative, andnegative for expression of Thy-1 (CD90).

The phrase “solid tumor” as used herein refers to an abnormal mass oftissue that usually does not contain cysts or liquid areas. Solid tumorsmay be benign or malignant. Different types of solid tumors are namedfor the type of cells that form them. Examples of solid tumors aresarcomas, carcinomas, lymphomas etc.

Anti-CD47 antibodies. Certain antibodies that bind CD47 prevent itsinteraction with SIRP□ receptor. Antibodies include free antibodies andantigen binding fragments derived therefrom, and conjugates, e.g.pegylated antibodies, drug, radioisotope, or toxin conjugates, and thelike.

Monoclonal antibodies directed against a specific epitope, orcombination of epitopes, will allow for the targeting and/or depletionof cellular populations expressing the marker. Various techniques can beutilized using monoclonal antibodies to screen for cellular populationsexpressing the marker(s), and include magnetic separation usingantibody-coated magnetic beads, “panning” with antibody attached to asolid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No.5,985,660; and Morrison et al. Cell, 96:737-49 (1999)). These techniquesallow for the screening of particular populations of cells; inimmunohistochemistry of biopsy samples; in detecting the presence ofmarkers shed by cancer cells into the blood and other biologic fluids,and the like.

Humanized versions of such antibodies are also within the scope of thisinvention. Humanized antibodies are especially useful for in vivoapplications in humans due to their low antigenicity.

The phrase “bispecific antibody” refers to a synthetic or recombinantantibody that recognizes more than one protein. Examples includebispecific antibodies 2B1, 520C9xH22, mDX-H210, and MDX447. Bispecificantibodies directed against a combination of epitopes, will allow forthe targeting and/or depletion of cellular populations expressing thecombination of epitopes. Exemplary bi-specific antibodies include thosetargeting a combination of CD47 and a cancer cell marker, such as, CD96,CD97, CD99, PTHR2, HAVCR2 etc. Generation of bi-specific antibody isdescribed in the literature, for example, in U.S. Pat. No. 5,989,830,U.S. Pat. No. 5,798,229, which are incorporated herein by reference.

The terms “treatment”, “treating”, “treat” and the like are used hereinto generally refer to obtaining a desired pharmacologic and/orphysiologic effect. The effect may be prophylactic in terms ofcompletely or partially preventing a disease or symptom thereof and/ormay be therapeutic in terms of a partial or complete stabilization orcure for a disease and/or adverse effect attributable to the disease.“Treatment” as used herein covers any treatment of a disease in amammal, particularly a human, and includes: (a) preventing the diseaseor symptom from occurring in a subject which may be predisposed to thedisease or symptom but has not yet been diagnosed as having it; (b)inhibiting the disease symptom, i.e., arresting its development; or (c)relieving the disease symptom, i.e., causing regression of the diseaseor symptom.

The terms “recipient”, “individual”, “subject”, “host”, and “patient”,used interchangeably herein and refer to any mammalian subject for whomdiagnosis, treatment, or therapy is desired, particularly humans.

A “host cell”, as used herein, refers to a microorganism or a eukaryoticcell or cell line cultured as a unicellular entity which can be, or hasbeen, used as a recipient for a recombinant vector or other transferpolynucleotides, and include the progeny of the original cell which hasbeen transfected. It is understood that the progeny of a single cell maynot necessarily be completely identical in morphology or in genomic ortotal DNA complement as the original parent, due to natural, accidental,or deliberate mutation.

The terms “cancer”, “neoplasm”, “tumor”, and “carcinoma”, are usedinterchangeably herein to refer to cells which exhibit relativelyautonomous growth, so that they exhibit an aberrant growth phenotypecharacterized by a significant loss of control of cell proliferation. Ingeneral, cells of interest for detection or treatment in the presentapplication include precancerous (e.g., benign), malignant,pre-metastatic, metastatic, and non-metastatic cells. Detection ofcancerous cells is of particular interest. The term “normal” as used inthe context of “normal cell,” is meant to refer to a cell of anuntransformed phenotype or exhibiting a morphology of a non-transformedcell of the tissue type being examined. “Cancerous phenotype” generallyrefers to any of a variety of biological phenomena that arecharacteristic of a cancerous cell, which phenomena can vary with thetype of cancer. The cancerous phenotype is generally identified byabnormalities in, for example, cell growth or proliferation (e.g.,uncontrolled growth or proliferation), regulation of the cell cycle,cell mobility, cell-cell interaction, or metastasis, etc.

Cancers of interest for treatment by the methods of the invention, e.g.treatment with an agent that binds to cell surface CD47 to increasephagocytosis of the cancer cells; include leukemias, particularly acuteleukemias such as T-ALL, B-ALL, AML, etc.; lymphomas (Hodgkins andnon-Hodgkins); sarcomas; melanomas; adenomas; carcinomas of solid tissueincluding ovarian carcinoma, breast carcinoma, pancreatic carcinoma,colon carcinoma, squamous cell carcinoma, transitional cell carcinoma,etc., hypoxic tumors, squamous cell carcinomas of the mouth, throat,larynx, and lung, genitourinary cancers such as cervical and bladdercancer, hematopoietic cancers, head and neck cancers, and nervous systemcancers, such as gliomas, astrocytomas, meningiomas, etc., benignlesions such as papillomas, and the like.

As used herein, a “target cell” is a cell expressing CD47 on thesurface, where masking or otherwise altering the CD47 positive phenotyperesults in altered phagocytosis. Usually a target cell is a mammaliancell, preferably a human cell.

A “biological sample” encompasses a variety of sample types obtainedfrom an individual and can be used in a diagnostic or monitoring assay.The definition encompasses blood and other liquid samples of biologicalorigin, solid tissue samples such as a biopsy specimen or tissuecultures or cells derived therefrom and the progeny thereof. Thedefinition also includes samples that have been manipulated in any wayafter their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such as proteinsor polynucleotides. The term “biological sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, celllysates, serum, plasma, biological fluid, and tissue samples.

An “individual” is a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, rodents, primates, farmanimals, sport animals, and pets.

An “effective amount” is an amount sufficient to effect beneficial ordesired clinical results. An effective amount can be administered in oneor more administrations. For purposes of this invention, an effectiveamount of a CD47 binding agent is an amount that is sufficient topalliate, ameliorate, stabilize, reverse, slow or delay the progressionof the disease state by modulating phagocytosis of a target cell.

As used herein, “treatment” is an approach for obtaining beneficial ordesired clinical results. For purposes of this invention, beneficial ordesired clinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, preventing spread (i.e., metastasis) ofdisease, delay or slowing of disease progression, amelioration orpalliation of the disease state, and remission (whether partial ortotal), whether detectable or undetectable. “Treatment” can also meanprolonging survival as compared to expected survival if not receivingtreatment. “Palliating” a disease means that the extent and/orundesirable clinical manifestations of a disease state are lessenedand/or time course of the progression is slowed or lengthened, ascompared to not administering the methods of the present invention.

“Therapeutic target” refers to a gene or gene product that, uponmodulation of its activity (e.g., by modulation of expression,biological activity, and the like), can provide for modulation of thecancerous phenotype. As used throughout, “modulation” is meant to referto an increase or a decrease in the indicated phenomenon (e.g.,modulation of a biological activity refers to an increase in abiological activity or a decrease in a biological activity).

Methods for Transplantation

Methods are provided to manipulate phagocytosis of circulatinghematopoietic cells. In some embodiments of the invention thecirculating cells are hematopoietic stem cells, or hematopoieticprogenitor cells, particularly in a transplantation context, whereprotection from phagocytosis is desirable. In other embodiments thecirculating cells are leukemia cells, particularly acute myeloidleukemia (AML), where increased phagocytosis is desirable.

In some embodiments of the invention, hematopoietic stem or progenitorcells are protected from phagocytosis in circulation by providing a hostanimal with a CD47 mimetic molecule, which interacts with SIRP□ onmacrophages and decreases macrophage phagocytosis. The CD47 mimetic maybe soluble CD47; CD47 coated on the surface of the cells to beprotected, a CD47 mimetic that binds to SIRP□ at the CD47 binding site,and the like. In some embodiments of the invention, CD47 is provided asa fusion protein, for example soluble CD47 fused to an Fc fragment,e.g., IgG1 Fc, IgG2 Fc, Ig A Fc etc.

Methods for generating proteins lacking the transmembrane region arewell known in the art. For example, a soluble CD47 can be generated byintroducing a stop codon immediately before the polynucleotide sequenceencoding the transmembrane region. Alternatively, the polynucleotidesequence encoding the transmembrane region can be replaced by apolynucleotide sequence encoding a fusion protein such as IgG1 Fc.Sequence for Fc fragments from different sources are available viapublicly accessible database including Entrez, Embl, etc. For example,mRNA encoding human IgG1 Fc fragment is provided by accession numberX70421.

The subject invention provide for methods for transplantinghematopoietic stem or progenitor cells into a mammalian recipient. Aneed for transplantation may be caused by genetic or environmentalconditions, e.g. chemotherapy, exposure to radiation, etc. The cells fortransplantation may be mixtures of cells, e.g. buffy coat lymphocytesfrom a donor, or may be partially or substantially pure. The cells maybe autologous cells, particularly if removed prior to cytoreductive orother therapy, or allogeneic cells, and may be used for hematopoieticstem or progenitor cell isolation and subsequent transplantation.

The cells may be combined with the soluble CD47 mimetic prior toadministration. For example, the cells may be combined with the mimeticat a concentration of from about 10 □g/ml, about 100 □g/ml, about 1mg/ml, about 10 mg/ml, etc., at a temperature of from about 4°, about10°, about 25° about 37°, for a period of time sufficient to coat thecells, where in some embodiments the cells are maintained on ice. Inother embodiments the cells are contacted with the CD47 mimeticimmediately prior to introduction into the recipient, where theconcentrations of mimetic are as described above.

The composition comprising hematopoietic stem or progenitor cells and aCD47 mimetic is administered in any physiologically acceptable medium,normally intravascularly, although they may also be introduced into boneor other convenient site, where the cells may find an appropriate sitefor regeneration and differentiation. Usually, at least 1×10⁵ cells willbe administered, preferably 1×10⁶ or more. The composition may beintroduced by injection, catheter, or the like.

Myeloproliferative Disorders, Leukemias, and Myelodysplastic Syndrome

Acute leukemias are rapidly progressing leukemia characterized byreplacement of normal bone marrow by blast cells of a clone arising frommalignant transformation of a hematopoietic cell. The acute leukemiasinclude acute lymphoblastic leukemia (ALL) and acute myelogenousleukemia (AML). ALL often involves the CNS, whereas acute monoblasticleukemia involves the gums, and AML involves localized collections inany site (granulocytic sarcomas or chloromas). In addition to CD47, wehave discovered a number of markers specific to AML SC. These includeCD96, CD97, CD99, PTHR2, HAVCR2 etc. These markers have been disclosedin U.S. Patent Application No. 61/011,324, filed on Jan. 15, 2008 andare hereby incorporated by reference.

The presenting symptoms are usually nonspecific (e.g., fatigue, fever,malaise, weight loss) and reflect the failure of normal hematopoiesis.Anemia and thrombocytopenia are very common (75 to 90%). The WBC countmay be decreased, normal, or increased. Blast cells are usually found inthe blood smear unless the WBC count is markedly decreased. The blastsof ALL can be distinguished from those of AML by histochemical studies,cytogenetics, immunophenotyping, and molecular biology studies. Inaddition to smears with the usual stains, terminal transferase,myeloperoxidase, Sudan black B, and specific and nonspecific esterase.

ALL is the most common malignancy in children, with a peak incidencefrom ages 3 to 5 yr. It also occurs in adolescents and has a second,lower peak in adults. Typical treatment emphasizes early introduction ofan intensive multidrug regimen, which may include prednisone,vincristine, anthracycline or asparaginase. Other drugs and combinationsare cytarabine and etoposide, and cyclophosphamide. Relapse usuallyoccurs in the bone marrow but may also occur in the CNS or testes, aloneor concurrent with bone marrow. Although second remissions can beinduced in many children, subsequent remissions tend to be brief.

The incidence of AML increases with age; it is the more common acuteleukemia in adults. AML may be associated with chemotherapy orirradiation (secondary AML). Remission induction rates are lower thanwith ALL, and long-term disease-free survival reportedly occurs in only20 to 40% of patients. Treatment differs most from ALL in that AMLresponds to fewer drugs. The basic induction regimen includescytarabine; along with daunorubicin or idarubicin. Some regimens include6-thioguanine, etoposide, vincristine, and prednisone.

Polycythemia vera (PV) is an idiopathic chronic myeloproliferativedisorder characterized by an increase in Hb concentration and RBC mass(erythrocytosis). PV occurs in about 2.3/100,000 people per year; moreoften in males (about 1.4:1). The mean age at diagnosis is 60 yr (range,15 to 90 yr; rarely in childhood); 5% of patients are <40 yr at onset.The bone marrow sometimes appears normal but usually is hypercellular;hyperplasia involves all marrow elements and replaces marrow fat. Thereis increased production and turnover of RBCs, neutrophils, andplatelets. Increased megakaryocytes may be present in clumps. Marrowiron is absent in >90% of patients, even when phlebotomy has not beenperformed.

Studies of women with PV who are heterozygous at the X-chromosome-linkedlocus for G6PD have shown that RBCs, neutrophils, and platelets have thesame G6PD isoenzyme, supporting a clonal origin of this disorder at apluripotent stem cell level.

Eventually, about 25% of patients have reduced RBC survival and fail toadequately increase erythropoiesis; anemia and myelofibrosis develop.Extramedullary hemopoiesis occurs in the spleen, liver, and other siteswith the potential for blood cell formation.

Without treatment, 50% of symptomatic patients die within 18 mo ofdiagnosis. With treatment, median survival is 7 to 15 yr. Thrombosis isthe most common cause of death, followed by complications of myeloidmetaplasia, hemorrhage, and development of leukemia.

The incidence of transformation into an acute leukemia is greater inpatients treated with radioactive phosphate (³²P) or alkylating agentsthan in those treated with phlebotomy alone. PV that transforms intoacute leukemia is more resistant to induction chemotherapy than de novoleukemia.

Because PV is the only form of erythrocytosis for which myelosuppressivetherapy may be indicated, accurate diagnosis is critical. Therapy mustbe individualized according to age, sex, medical status, clinicalmanifestations, and hematologic findings.

Myelodysplastic syndrome (MDS) is a group of syndromes (preleukemia,refractory anemias, Ph-negative chronic myelocytic leukemia, chronicmyelomonocytic leukemia, myeloid metaplasia) commonly seen in olderpatients. Exposure to carcinogens may by be implicated. MDS ischaracterized by clonal proliferation of hematopoietic cells, includingerythroid, myeloid, and megakaryocytic forms. The bone marrow is normalor hypercellular, and ineffective hematopoiesis causes variablecytopenias, the most frequent being anemia. The disordered cellproduction is also associated with morphologic cellular abnormalities inmarrow and blood. Extramedullary hematopoiesis may occur, leading tohepatomegaly and splenomegaly. Myelofibrosis is occasionally present atdiagnosis or may develop during the course of MDS. The MDS clone isunstable and tends to progress to AML.

Anemia is the most common clinical feature, associated usually withmacrocytosis and anisocytosis. Some degree of thrombocytopenia is usual;on blood smear, the platelets vary in size, and some appearhypogranular. The WBC count may be normal, increased, or decreased.Neutrophil cytoplasmic granularity is abnormal, with anisocytosis andvariable numbers of granules. Eosinophils also may have abnormalgranularity. A monocytosis is characteristic of the chronicmyelomonocytic leukemia subgroup, and immature myeloid cells may occurin the less well differentiated subgroups. The prognosis is highlydependent on classification and on any associated disease. Response ofMDS to AML chemotherapy is similar to that of AML, after age andkaryotype are considered.

Treatment of Cancer

The invention provides methods for reducing growth of cancer cells byincreasing their clearance by phagocytosis, through the introduction ofa CD47 blocking agent, e.g. an anti-CD47 antibody. In certainembodiments the cancer cells may be AML stem cells. In otherembodiments, the cancer cells may be those of a solid tumor, such ascarcinomas, glioblastoma, melanoma, etc. By blocking the activity ofCD47, the downregulation of phagocytosis that is found with certaintumor cells, e.g. AML cells, is prevented.

“Reducing growth of cancer cells” includes, but is not limited to,reducing proliferation of cancer cells, and reducing the incidence of anon-cancerous cell becoming a cancerous cell. Whether a reduction incancer cell growth has been achieved can be readily determined using anyknown assay, including, but not limited to, [³H]-thymidineincorporation; counting cell number over a period of time; detectingand/or measuring a marker associated with AML, etc.

Whether a substance, or a specific amount of the substance, is effectivein treating cancer can be assessed using any of a variety of knowndiagnostic assays for cancer, including, but not limited to biopsy,contrast radiographic studies, CAT scan, and detection of a tumor markerassociated with cancer in the blood of the individual. The substance canbe administered systemically or locally, usually systemically.

As an alternative embodiment, an agent, e.g. a chemotherapeutic drugthat reduces cancer cell growth, can be targeted to a cancer cell byconjugation to a CD47 specific antibody. Thus, in some embodiments, theinvention provides a method of delivering a drug to a cancer cell,comprising administering a drug-antibody complex to a subject, whereinthe antibody is specific for a cancer-associated polypeptide, and thedrug is one that reduces cancer cell growth, a variety of which areknown in the art. Targeting can be accomplished by coupling (e.g.,linking, directly or via a linker molecule, either covalently ornon-covalently, so as to form a drug-antibody complex) a drug to anantibody specific for a cancer-associated polypeptide. Methods ofcoupling a drug to an antibody are well known in the art and need not beelaborated upon herein.

In certain embodiments, a bi-specific antibody may be used. For examplea bi-specific antibody in which one antigen binding domain is directedagainst CD47 and the other antigen binding domain is directed against acancer cell marker, such as, CD96 CD97, CD99, PTHR2, HAVCR2 etc. may beused.

Depletion of AMLSC is useful in the treatment of AML. Depletion can beachieved by several methods. Depletion is defined as a reduction in thetarget population by up to about 30%, or up to about 40%, or up to about50%, or up to about 75% or more. An effective depletion is usuallydetermined by the sensitivity of the particular disease condition to thelevels of the target population. Thus in the treatment of certainconditions a depletion of even about 20% could be beneficial.

A CD47 specific agent that specifically depletes the targeted AMLSC isused to contact the patient blood in vitro or in vivo, wherein after thecontacting step, there is a reduction in the number of viable AMLSC inthe targeted population. An effective dose of antibodies for such apurpose is sufficient to decrease the targeted population to the desiredlevel, for example as described above. Antibodies for such purposes mayhave low antigenicity in humans or may be humanized antibodies.

In one embodiment of the invention, antibodies for depleting targetpopulation are added to patient blood in vivo. In another embodiment,the antibodies are added to the patient blood ex vivo. Beads coated withthe antibody of interest can be added to the blood, target cells boundto these beads can then be removed from the blood using procedurescommon in the art. In one embodiment the beads are magnetic and areremoved using a magnet. Alternatively, when the antibody isbiotinylated, it is also possible to indirectly immobilize the antibodyonto a solid phase which has adsorbed avidin, streptavidin, or the like.The solid phase, usually agarose or sepharose beads are separated fromthe blood by brief centrifugation. Multiple methods for taggingantibodies and removing such antibodies and any cells bound to theantibodies are routine in the art. Once the desired degree of depletionhas been achieved, the blood is returned to the patient. Depletion oftarget cells ex vivo decreases the side effects such as infusionreactions associated with the intravenous administration. An additionaladvantage is that the repertoire of available antibodies is expandedsignificantly as this procedure does not have to be limited toantibodies with low antigenicity in humans or humanized antibodies.

Example 1 CD47 is a Marker of Myeloid Leukemias

Materials and Methods

Immunohistochemistry.

Cytospins of double sorted myeloid progenitor populations (CMP, GMP),IL-3R□ high CD45 RA+ cells and CD14+c-kit+ lin− cells were performedusing a Shandon cytospin apparatus. Cytospins were stained with Giemsadiluted 1/5 with H20 for 10 min followed by staining with May-Grunwaldfor 20 minutes. Cytospins were analyzed with the aid of a Zeissmicroscope.

Human Bone Marrow and Peripheral Blood Samples.

Normal bone marrow samples were obtained with informed consent from20-25 year old paid donors who were hepatitis A, B, C and HIV negativeby serology (All Cells). CMML bone marrow samples were obtained withinformed consent, from previously untreated patients, at StanfordUniversity Medical Center.

Human Bone Marrow HSC and Myeloid Progenitor Flow-Cytometric Analysisand Cell Sorting.

Mononuclear fractions were extracted following Ficoll densitycentrifugation according to standard methods and analyzed fresh orsubsequent to rapid thawing of samples previously frozen in 90% FCS and10% DMSO in liquid nitrogen. In some cases, CD34+ cells were enrichedfrom mononuclear fractions with the aid of immunomagnetic beads (CD34+Progenitor Isolation Kit, Miltenyi Biotec, Bergisch-Gladbach, Germany).Prior to FACS analysis and sorting, myeloid progenitors were stainedwith lineage marker specific phycoerythrin (PE)-Cy5-conjugatedantibodies including CD2 RPA-2.10; CD11b, ICRF44; CD20, 2H7; CD56, B159;GPA, GA-R2 (Becton Dickinson—PharMingen, San Diego), CD3, S4.1; CD4,S3.5; CD7, CD7-6B7; CD8, 3B5; CD10, 5-1B4, CD14, TUK4; CD19, SJ25-C1(Caltag, South San Francisco, Calif.) and APC-conjugated anti-CD34,HPCA-2 (Becton Dickinson-PharMingen), biotinylated anti-CD38, HIT2(Caltag) in addition to PE-conjugated anti-IL-3R□, 9F5 (BectonDickinson-ParMingen) and FITC-conjugated anti-CD45RA, MEM56 (Caltag)followed by staining with Streptavidin—Texas Red to visualize CD38-BIOstained cells and resuspension in propidium iodide to exclude deadcells. Unstained samples and isotype controls were included to assessbackground fluorescence.

Following staining, cells were analyzed and sorted using a modified FACSVantage (Becton Dickinson Immunocytometry Systems, Mountain View,Calif.) equipped with a 599 nm dye laser and a 488 nm argon laser.Double sorted progenitor cells (HSC) were identified as CD34+CD38+ andlineage negative. Common myeloid progenitors (CMP) were identified basedon CD34+CD38+IL-3R□+CD45RA− lin− staining and their progeny includinggranulocyte/macrophage progenitors (GMP) were CD34+CD38+IL-3R□+CD45RA+while megakaryocyte/erythrocyte progenitors (MEP) were identified basedon CD34+CD38+IL-3R□− CD45RA− lin− staining (Manz, PNAS 11872).

CD47 Expression by Normal Versus Myeloproliferative and AML Progenitors

Peripheral blood and bone marrow samples were obtained with informedconsent from patients with myeloproliferative disorders and acutemyelogenous leukemia at Stanford University Medical Center according toStanford IRB and HIPAA regulations. Peripheral blood or bone marrowmononuclear cells (1-5×10⁶ cells) were stained with lineage cocktail asabove but excluding CD7, CD11b and CD14. Subsequently, samples werestained with CD14 PE (1/25), CD47 FITC (1/25), CD38 Bio (Bio) and c-kitAPC (1/25) or CD34 APC or FITC (1/50) for 45 min followed by washing andstaining with Streptavidin Texas Red (1/25) for 45 min and finallyresuspension in propidium iodide.

DISCUSSION

Here we show that CD47 overexpression is characteristic of progressionof human myeloproliferative disorders to AML (see FIGS. 1-5B). CD47controls integrin function but also the ability of macrophages tophagocytose cells, depending on the level of CD47 expression. Thus,aberrant CD47 expression may allow LSC to evade both innate and adaptivehost immunity.

Human CD47 expression analysis was performed via FACS on human normal,pre-leukemic myeloproliferative disorder (MPD) or AML HSC, progenitorsand lineage positive cells derived from marrow or peripheral blood. MPDsamples (n=63) included polycythemia vera (PV; n=15), post-polycythemicmyeloid metaplasia/myelofibrosis (PPMM/MF; n=5), essentialthrombocythemia (ET; n=8), atypical chronic myelogenous leukemia (aCML;n=2), CML (n=7), chronic eosinophilic leukemia (CEL; n=1), chronicmyelomonocytic leukemia (CMML; n=13) and acute myelogenous leukemia(AML; n=12). As we have observed with the transgenic leukemic mousemodels, progression of human myeloproliferative disorders to AML (n=12)was associated with an expansion of the GMP pool (70.6%+/−S.D. 2.15)compared with normal bone marrow (14.7%+/−S.D. 2.3). Furthermore, FACSanalysis revealed that CD47 expression first increased 1.7 fold in AMLcompared with normal HSC and then increased to 2.2 fold greater thannormal with commitment of AML progenitors to the myeloid lineage. CD47was over-expressed by AML primitive progenitors and their progeny butnot by the majority of MPD (MFI 2.3+/−S.D. 0.43) compared with normalbone marrow (MFI 1.9+/−S.D. 0.07). Thus, increased CD47 expression is auseful diagnostic marker for progression to AML and in additionrepresents a novel therapeutic target.

Example 2 Human and Mouse Leukemias Upregulate CD47 to Evade MacrophageKilling

iCD47 Facilitates Engraftment, Inhibits Phagocytosis, and is More HighlyExpressed on AML LSC.

We determined expression of CD47 on human AML LSC and normal HSC by flowcytometry. HSC (Lin−CD34+CD38−CD90+) from three samples of normal humanmobilized peripheral blood and AML LSC (Lin−CD34+CD38−CD90−) from sevensamples of human AML were analyzed for surface expression of CD47 (FIG.6). CD47 was expressed at low levels on the surface of normal HSC;however, on average, it was approximately 5-fold more highly expressedon AML LSC, as well as bulk leukemic blasts.

Anti-Human CD47 Monoclonal Antibody Stimulates Phagocytosis and InhibitsEngraftment of AML LSC.

In order to test the model that CD47 overexpression on AML LSC preventsphagocytosis of these cells through its interaction with SIRP□□□ oneffector cells, we have utilized a monoclonal antibody directed againstCD47 known to disrupt the CD47-SIRP□□□ interaction. The hybridomaproducing a mouse-anti-human CD47 monoclonal antibody, termed B6H12, wasobtained from ATCC and used to produce purified antibody. First, weconducted in vitro phagocytosis assays. Primary human AML LSC werepurified by FACS from two samples of human AML, and then loaded with thefluorescent dye CFSE. These cells were incubated with mouse bonemarrow-derived macrophages and monitored using immunofluorescencemicroscopy (FIG. 7) and flow cytometry (FIG. 9) to identify phagocytosedcells. In both cases, no phagocytosis was observed in the presence of anisotype control antibody; however, significant phagocytosis was detectedwith the addition of the anti-CD47 antibody (FIG. 9). Thus, blockage ofhuman CD47 with a monoclonal antibody is capable of stimulating thephagocytosis of these cells by mouse macrophages.

We next investigated the ability of the anti-CD47 antibody to inhibitAML LSC engraftment in vivo. Two primary human AML samples were eitheruntreated or coated with the anti-CD47 antibody prior to transplantationinto NOG newborn mice. 13 weeks later, the mice were sacrificed andanalyzed for human leukemia bone marrow engraftment by flow cytometry(FIG. 10). The control mice demonstrated leukemic engraftment while micetransplanted with the anti-CD47-coated cells showed little to noengraftment. These data indicate that blockade of human CD47 with amonoclonal antibody is able to inhibit AML LSC engraftment.

CD96 is a Human Acute Myeloid Leukemia Stem Cell-Specific Cell SurfaceMolecule.

CD96, originally termed Tactile, was first identified as a T cellsurface molecule that is highly upregulated upon T cell activation. CD96is expressed at low levels on resting T and NK cells and is stronglyupregulated upon stimulation in both cell types. It is not expressed onother hematopoietic cells, and examination of its expression patternshowed that it is only otherwise present on some intestinal epithelia.The cytoplasmic domain of CD96 contains a putative ITIM motif, but it isnot know if this functions in signal transduction. CD96 promotesadhesion of NK cells to target cells expressing CD155, resulting instimulation of cytotoxicity of activated NK cells.

Preferential Cell Surface Expression of Molecules Identified from GeneExpression Analysis.

Beyond CD47 and CD96, several molecules described in U.S. PatentApplication No. 61/011,324 are known to be expressed on AML LSC,including: CD123, CD44, CD99 and CD33.

Tumor progression is characterized by several hallmarks, includinggrowth signal independence, inhibition of apoptosis, and evasion of theimmune system, among others. We show here that expression of CD47, aligand for the macrophage inhibitory signal regulatory protein alpha(SIRPα) receptor, is increased in human and mouse myeloid leukaemia andallows cells to evade phagocytosis and increase their tumorigenicpotential. CD47, also known as integrin associated protein (IAP), is animmunoglobulin-like transmembrane pentaspanin that is broadly expressedin mammalian tissues. We provide evidence that CD47 is upregulated inmouse and human myeloid leukaemia stem and progenitor cells, as well asleukemic blasts. Consistent with a biological role for CD47 in myeloidleukaemia development and maintenance, we demonstrate that ectopicover-expression of CD47 allows a myeloid leukaemia cell line to grow inmice that are T, B, and NK-cell deficient, whereas it is otherwisecleared rapidly when transplanted into these recipients. Theleukemogenic potential of CD47 is also shown to be dose-dependent, ashigher expressing clones have greater tumor forming potential than lowerexpressing clones. We also show that CD47 functions in promotingleukemogenesis by inhibiting phagocytosis of the leukemic cells bymacrophages.

CD47 is significantly upregulated in leukemic Fas^(lpr/lpr)×hMRP8bcl2transgenic bone marrow, and in leukemic hMRP8bcr/abl×hMRP8bcl2 mice.Transcripts for CD47 are increased in leukemic hMRP8bcr/abl×hMRP8bcl2bone marrow 3-4 fold by quantitative RT-PCR and 6-7 fold in c-Kitenriched leukemic marrow relative to healthy hMRP8bcl2+ bone marrow(FIG. Ile). Leukemic spleen had an expansion of the granulocytemacrophage progenitor (GMP) population as well as c-Kit+ Sca-1+ Lin-stemand progenitor subsets relative to control mice, which were of the samegenotype as leukemic mice but failed to develop disease (FIG. 11a-d ).Expression levels for CD47 protein were found to begin increasing inleukemic mice relative to control mice at the stage of theFlk2−CD34−c-Kit+ Sca-1+ Lin− long-term hematopoietic stem cell (LT-HSC)(FIG. 11f ). This increased level of expression was maintained in GMPand Mac-1+ blasts, but not megakaryocyte/erythroid restrictedprogenitors (MEP) (FIG. 11f ). The increase in CD47 between leukemic andnormal cells was between 3 to 20 fold. All mice that developed leukaemiathat we have examined from hMRP8bcr/abl×hMRP8bcl2 primary (n=3) andsecondary transplanted mice (n=3), Fas^(lpr/lpr)×hMRP8bcl2 primary(n=14) and secondary (n=19) mice, and hMRP8bcl2×hMRP8bcl2 primary (n=3)and secondary (n=12) mice had increased CD47 expression. We have alsofound increased CD47 expression in mice that received p210bcr/ablretrovirally-transduced mouse bone marrow cells that developed leukemia.

FACS-mediated analysis of human hematopoietic progenitor populations wasperformed on blood and marrow derived from normal cord blood andmobilized peripheral blood (n=16) and myeloproliferative disorders(MPDS) including polycythemia vera (PV; n=16), myelofibrosis (MF; n=5),essential thrombocythemia (ET; n=7), chronic myelomonocytic leukaemia(CMML; n=11) and atypical chronic myeloid leukaemia (aCML; n=1) as wellas blast crisis phase chronic myeloid leukaemia (CML; n=19), chronicphase CIVIL (n=7) and acute myelogenous leukaemia (AML; n=13). Thisanalysis demonstrated that granulocyte-macrophage progenitors (GMP)expanded in MPDS with myeloid skewed differentiation potential includingatypical CIVIL, proliferative phase CMML and acute leukaemia includingblast crisis CIVIL and AML (FIG. 12a ). AML HSC and progenitorsuniformly exhibited higher levels of CD47 expression compared withnormal controls (FIG. 12b ); every sample from BC-CML and AML hadelevated levels of CD47. Moreover, progression from chronic phase CIVILto blast crisis was associated with a significant increase in CD47expression (FIG. 12c ). Using the methods described in this study, wehave found that human CD47 protein expression in CIVIL-BC increased 2.2fold in CD90+ CD34+ CD38− Lin− cells relative to normal (p=6.3×10⁻⁵),2.3 fold in CD90− CD34+ CD38− Lin− cells relative to normal(p=4.3×10⁻⁵), and 2.4 fold in CD 34+CD38+ Lin− cells (p=′7.6×10⁻⁶)(FIGS. 12b-12c ); however, using a newer optimized staining protocol wehave observed that CD47 is increased approximately 10 fold in AML andBC-CIVIL compared to normal human HSCs and progenitors.

It was then asked whether forced expression of mouse CD47 on humanleukemic cells would confer a competitive advantage in forming tumors inmice. MOLM-13 cells, which are derived from a patient with AML 5a, weretransduced with Tet-MCS-IRES-GFP (Tet) or Tet-CD47-MCS-IRES-GFP(Tet-CD47) (FIG. 13a ), and stable integrants were propagated on thebasis of GFP expression. The cells were then transplanted intravenouslyin a competitive setting with untransduced MOLM-13 cells into T, B, andNK deficient recombination activating gene 2, common gamma chaindeficient (RAG2−/−, Gc−/−) mice. Only cells transduced with Tet-CD47were able to give rise to tumors in these mice, efficiently engraftingbone marrow, spleen and peripheral blood (FIGS. 13a-b ). The tumors werealso characterized by large tumor burden in the liver (FIGS. 13b, 13g ),which is particularly significant because the liver is thought to havethe highest number of macrophages of any organ, with estimates thatKupffer cells may comprise 80% of the total tissue macrophagepopulation. These cells also make up 30% of the sinusoidal lining,thereby strategically placing them at sites of entry into the liver.Hence, significant engraftment there would have to disable a macrophagecytotoxic response. In addition to developing tumor nodules, theTet-CD47 MOLM-13 cells exhibited patterns of hepatic involvementtypically seen with human AML, with leukemic cells infiltrating theliver with a sinusoidal and perivenous pattern. (FIG. 13d ). Overall,Tet-CD47 MOLM-13 transplanted mice died more quickly than Tet MOLM-13transplanted mice, which had virtually no engraftment of leukemic cellsin hematopoietic tissues (FIG. 13c ). Tet-MOLM-13 mice still hadsignificant mortality, most likely due to localized growth at the siteof injection (retro-orbital sinus) with extension into the brain.

Since CD47 has been shown to be important for the migration ofhematopoietic cells, and is known to modulate binding to extracellularmatrix proteins, either by direct interaction or via its effect onintegrins, one possibility for the lack of growth of Tet MOLM-13 cellsin mice was their inability to migrate to niches. To test thispossibility, Tet MOLM-13 or Tet-CD47 MOLM-13 cells were directlyinjected into the femoral cavity of immunodeficient mice. Tet-CD47MOLM-13 cells were able to engraft all bones and other hematopoietictissues of recipient mice, whereas Tet MOLM-13 cells had minimal, ifany, engraftment only at the site of injection (FIG. 13e ). Micetransplanted in this manner with Tet-CD47 MOLM-13 cells died atapproximately 50-60 days post-transplant (n=4), whereas mice thatreceived Tet MOLM-13 (n=5) cells remained alive for at least 75 dayswithout signs of disease at which point they were euthanized foranalysis. These results suggest a function other than or in addition tomigration or homing for CD47 in MOLM-13 engraftment.

Complete lack of CD47 has been shown to result in phagocytosis oftransplanted murine erythrocytes and leukocytes, via lack of interactionwith SIRPα on macrophages. Thus, we tested whether over-expression ofCD47 could prevent phagocytosis of live, unopsonized MOLM-13 cells. Weincubated Tet or Tet-CD47 MOLM-13 cells with bone marrow derivedmacrophages (BMDM) for 2-24 hours and assessed phagocytosis by countingthe number of ingested GFP+ cells under a microscope or by evaluatingthe frequency of GFP+ macrophages using a flow cytometer. Expression ofCD47 dramatically lowered macrophage clearance of these cells at alltime points tested, whereas Tet-MOLM-13 were quickly phagocytosed in amanner that increased over time (FIGS. 14a-c ). We also injected MOLM-13cells into mice and analyzed hematopoietic organs 2 hours later forevidence of macrophage phagocytosis. Macrophages in bone marrow, spleen,and liver all had higher GFP+ fraction when injected with Tet MOLM-13cells as compared to CD47 expressing cells. This indicates that CD47over-expression can compensate for pro-phagocytic signals alreadypresent on leukemic cells, allowing them to survive when they wouldotherwise be cleared by macrophages.

Recent report indicates that lack of CD47 reactivity across speciesmight mediate xenorejections of transplanted cells. Furthermore, arecent study has demonstrated that human CD47 is unable to interact withSIRPα from C57Bl/6 mice, but is able to react with receptor fromnon-obese diabetic (NOD) mice, which are more permissive for human cellengraftment than C57Bl/6 mice. Furthermore, we have also observed thatHL-60 cells, a human promyelocytic cell line with higher levels of humanCD47 expression than MOLM-13, are able to engraft mice and causeleukaemia. Jurkat cells, a human T-lymphocyte cell line, are very highfor human CD47 and are phagocytosed by murine macrophages in vitro at amuch lower rate than MOLM-13. Thus, our data indicate that the abilityof cells to engraft mice in vivo or evade phagocytosis in vitro by mousemacrophages correlates with the level of human CD47 expression.

To model the tumorigenic effect of having high versus low CD47expression, we sorted clones of murine CD47 expressing MOLM-13 cellsinto high and low expressers. When adjusted for cell size, CD47 densityon the CD47^(lo) MOLM-13 cells was approximately equal to mouse bonemarrow cells, whereas CD47^(hi) MOLM-13 cells had approximately 9 foldhigher expression, an increase commensurate with the change seen in CD47expression on primary leukemic cells compared to their normalcounterparts (FIG. 15a ). When high or low expressing cells weretransplanted into recipients, only mice transplanted with highexpressing cells succumbed to disease by 75 days of age (FIG. 15c ).Furthermore, organomegaly was more pronounced in mice transplanted withhigh expressing cells (FIG. 15d ). Mice receiving CD47^(lo) MOLM-13cells still had notable liver masses. However, the masses wereinvariably 1-2 large nodes that were well-encapsulated and physicallysegregated from the liver parenchyma, in marked contrast to tumor massesfrom CD47hi MOLM-13 cells which consisted of hundreds of small massesscattered throughout the parenchyma. Thus, these large tumor massesconsist of cells which have found macrophage free-niches to grow inseparate from the main organ body. As expected, the infiltration ofMOLM-13 cells in bone marrow and spleen of recipient mice was muchhigher for mice transplanted with CD47^(hi) MOLM-13 cells as well (FIG.15e ). We also examined the level of CD47 expression in two mice thatreceived CD47^(lo) MOLM-13 cells but had significant marrow engraftment.In both cases, the persisting cells after 75 days had much higher levelsof CD47 than the original line (FIG. 15f ), indicating that a strongselection pressure exists in vivo for high levels of CD47 expression onleukemic cells. In total, these data indicate that CD47 expression levelis a significant factor in tumorigenic potential, and that in aheterogeneous population of leukemic cells, strong selection exists forthose clones with high CD47 expression.

We then asked if higher CD47 expression level would provide addedprotection against macrophage phagocytosis. We performed an in vitrophagocytosis assay with CD47^(hi) and CD47^(lo) MOLM-13 red fluorescentprotein (RFP) expressing cells. After incubation with macrophages, fargreater numbers of CD47^(lo) cells were phagocytosed as compared toCD47^(hi) cells (FIG. 15g ). If phagocytic indices are compared forcontrol MOLM-13 cells, bulk (un-sorted) CD47 MOLM-13 cells, CD47^(lo),and CD47^(hi) MOLM-13 cells, then a direct correlation between CD47expression level and ability to evade phagocytosis can be seen (FIG. 14a, FIG. 15f ). Furthermore, when CD47^(lo) RFP MOLM-13 cells andCD47^(hi) GFP MOLM-13 cells were co-incubated with macrophages in thesame wells, the low expressing cells were far more likely to bephagocytosed (FIG. 15h, 15i ). Thus, in a mixed population of cells withvarying levels of CD47 expression, the low expressing cells are morelikely to be cleared by phagocytic clearance over time.

We also titrated CD47 expression using another method. Since CD47 isexpressed in MOLM-13 cells using a Tet-OFF system, we utilized theTet-inducible promoter element to control expression of CD47 in MOLM-13cells. Beginning two weeks after transplantation with CD47^(hi) MOLM-13cells, a cohort of mice was given doxycycline and followed for up to 75days post-transplant. During this time course, none of the mice givendoxycycline succumbed to disease or had large infiltration of MOLM-13cells in hematopoietic organs (FIGS. 15b-d ). At the doses ofdoxycycline used in this experiment, muCD47 expression in MOLM-13 cellswas reduced to levels below that of normal mouse bone marrow, butnotably not completely absent (FIG. 15b ). Thus, a sustained high levelof CD47 expression is required for robust MOLM-13 survival inhematopoietic organs.

Many examples of tumor clearance by T, B, and NK cells have beendescribed in the literature, indicating that a healthy immune system isessential for regulating nascent tumor growth. However, to date, fewexamples have been produced indicating that macrophage-mediatedphagocytosis can check tumor development. Collectively, our studiesreveal that ectopic expression of CD47 can enable otherwise immunogenictumor cells to grow rapidly in a T, B, and NK-cell deficient host.Furthermore, this is likely to reflect a mechanism used by human myeloidleukemias to evade the host immune system since CD47 is consistentlyupregulated in murine and human myeloid leukemias, including all formsof chronic and acute myeloid leukaemia tested thus far. Thus, it appearslikely that tumor cells are capable of being recognized as a target byactivated macrophages and cleared through phagocytosis. By upregulatingCD47, cancers are able to escape this form of innate immune tumorsurveillance.

This form of immune evasion is particularly important since thesecancers often occupy sites of high macrophage infiltration. CD47 wasfirst cloned as an ovarian tumor cell marker, indicating that it mayplay a role in preventing phagocytosis of other tissue cancers as well.Furthermore, solid tumors often metastasize to macrophage rich tissuessuch as liver, lung, bone marrow, and lymph nodes, indicating that theymust be able to escape macrophage-mediated killing in those tissues.Finding methods to disrupt CD47-SIRPα interaction may thus prove broadlyuseful in developing novel therapies for cancer. Preventing CD47-SIRPαinteraction is doubly effective since antigens from phagocytosed tumorcells may be presented by macrophages to activate an adaptive immuneresponse, leading to further tumor destruction.

Methods

Mice.

hMRP8bcrabl, hMRP8bcl2, and Fas^(lpr/lpr) transgenic mice were createdas previously described and crossed to obtain double transgenics.hMRP8bcl2 homozygotes were obtained by crossing heterozygote mice toeach other. C57Bl/6 Ka mice from our colony were used as a source ofwild-type cells. For transplant experiments, cells were transplantedinto C57Bl/6 RAG2^(−/−) common gamma chain (Gc)^(−/−) mice given aradiation dose of 4 Gy using gamma rays from a cesium irradiator(Phillips). Primary mouse leukemias were transplanted into CD45.2C57Bl6/Ka mice given a radiation dose of 9.5 Gy. Mice were euthanizedwhen moribund.

Mouse Tissues.

Long bones were flushed with PBS supplemented with 2% fetal calf serumstaining media (SM) Spleens and livers were dissociated using frostedglass slides in SM, then passed through a nylon mesh. All samples weretreated with ACK lysis buffer to lyse erythrocytes prior to furtheranalysis.

Quantitative RT-PCR Analysis.

Bone marrow was obtained from leukemic hMRP8bcr/abl×hMRP8bcl2 mice orhMRP8bcl2 control mice. Cells were c-Kit enriched using c-Kit microbeadsand an autoMACS column (Miltenyi). RNA was extracted using Trizolreagent (Invitrogen) and reverse transcription performed usingSuperScriptII reverse polymerase (Invitrogen). cDNA corresponding toapproximately 1000 cells was used per PCR reaction. Quantitative PCR wasperformed with a SYBR green kit on an ABI Prism 7000 PCR (AppliedBiosystems) machine at 50° C. for 2 minutes, followed by 95° C. for 10minutes and then 40 cycles of 95° C. for 15 minutes followed by 60° C.for 1 minute. Beta-actin and 18S RNA were used as controls for cDNAquantity and results of CD47 expression were normalized. Sequences for18S RNA forward and reverse primers were TTGACGGAAGGGCACCACCAG (SEQ IDNO: 8) and GCACCACCACCCACGGAATCG (SEQ ID NO: 9), respectively, forbeta-actin were TTCCTTCTTGGGTATGGAAT (SEQ ID NO: 10) andGAGCAATGATCTTGATCCTC (SEQ ID NO: 11), and for CD47 wereAGGCCAAGTCCAGAAGCATTC (SEQ ID NO: 12) and AATCATTCTGCTGCTCGTTGC (SEQ IDNO: 13).

Human Bone Marrow and Peripheral Blood Samples.

Normal bone marrow samples were obtained with informed consent from20-25 year old paid donors who were hepatitis A, B, C and HIV negativeby serology (All Cells). Blood and marrow cells were donated by patientswith chronic myelomonocytic leukemia (CMML), chronic myeloid leukemia(CML), and acute myelogenous leukemia (AML) and were obtained withinformed consent, from previously untreated patients.

Cell Lines.

MOLM-13 cells were obtained from DSMZ. HL-60 and Jurkat cells wereobtained from ATCC. Cells were maintained in Iscove's modifiedDulbecco's media (IMDM) plus 10% fetal bovine serum (FBS) (Hyclone). Tofractionate MOLM-13 cells into those with high and low CD47 expression,Tet-CD47 MOLM-13 cells were stained with anti-mouse CD47 Alexa-680antibody (mIAP301). The highest and lowest 5% of mouse CD47 expressingcells was sorted on a BD FACSAria and re-grown in IMDM+10% FCS for 2weeks. The cells were sorted for three more rounds of selectionfollowing the same protocol to obtain the high and low expressing cellsused in this study. To obtain red fluorescent protein (RFP) constructs,the mCherry RFP DNA was cloned into Lentilox 3.7 (pLL3.7) empty vector.Lentivirus obtained from this construct was then used to infect celllines.

Cell Staining and Flow Cytometry.

Staining for mouse stem and progenitor cells was performed using thefollowing monoclonal antibodies: Mac-1, Gr-1, CD3, CD4, CD8, B220, andTer119 conjugated to Cy5-PE (eBioscience) were used in the lineagecocktail, c-Kit PE-Cy7 (eBioscience), Sca-1 Alexa680 (e13-161-7,produced in our lab), CD34 FITC (eBioscience), CD16/32(FcGRII/III) APC(Pharmingen), and CD135(Flk-2) PE (eBioscience) were used as previouslydescribed to stain mouse stem and progenitor subsets. Mouse CD47antibody (clone mIAP301) was assessed using biotinylated antibodyproduced in our lab. Cells were then stained with streptavidinconjugated Quantum Dot 605 (Chemicon). Samples were analyzed using aFACSAria (Beckton Dickinson).

For human samples, mononuclear fractions were extracted following Ficolldensity centrifugation according to standard methods and analyzed freshor subsequent to rapid thawing of samples previously frozen in 90% FCSand 10% DMSO in liquid nitrogen. In some cases, CD34+ cells wereenriched from mononuclear fractions with the aid of immunomagnetic beads(CD34+ Progenitor Isolation Kit, Miltenyi Biotec, Bergisch-Gladbach,Germany). Prior to FACS analysis and sorting, myeloid progenitors werestained with lineage marker specific phycoerythrin (PE)-Cy5-conjugatedantibodies including CD2 RPA-2.10; CD11b, ICRF44; CD20, 2H7; CD56, B159;GPA, GA-R2 (Becton Dickinson—PharMingen, San Diego), CD3, S4.1; CD4,S3.5; CD7, CD7-6B7; CD8, 3B5; CD10, 5-1B4, CD14, TUK4; CD19, SJ25-C1(Caltag, South San Francisco, Calif.) and APC-conjugated anti-CD34,HPCA-2 (Becton Dickinson-PharMingen), biotinylated anti-CD38, HIT2(Caltag) in addition to PE-conjugated anti-IL-3R□, 9F5 (BectonDickinson—ParMingen) and FITC-conjugated anti-CD45RA, MEM56 (Caltag)followed by staining with Streptavidin—Texas Red to visualize CD38-BIOstained cells.

Following staining, cells were analyzed using a modified FACS Vantage(Becton Dickinson Immunocytometry Systems, Mountain View, Calif.)equipped with a 599 nm dye laser and a 488 nm argon laser or a FACSAria.Hematopoietic stem cells (HSC) were identified as CD34+CD38+CD90+ andlineage negative. Anti-human CD47 FITC (clone B6H12, Pharmingen) wasused to assess CD47 expression in all human samples. Fold change forCD47 expression was determined by dividing the average mean fluorescenceintensity of CD47 for all the samples of CML-BC, CML-CP, or AML by theaverage mean fluorescence intensity of normal cells for a given cellpopulation. Common myeloid progenitors (CMP) were identified based onCD34+CD38+IL-3R□+CD45RA− lin− staining and their progeny includinggranulocyte/macrophage progenitors (GMP) wereCD34+CD38+IL-3R□+CD45RA+Lin− while megakaryocyte/erythrocyte progenitors(MEP) were identified based on CD34+CD38+IL-3R□− CD45RA− Lin− staining.

To determine the density of mouse or human CD47, cells were stained withsaturating amounts of anti-CD47 antibody and analyzed on a FACSAria.Since forward scatter is directly proportional to cell diameter, anddensity is equal to expression level per unit of surface area we usedFloJo software to calculate geometric mean fluorescent intensity of theCD47 channel and divided by the geometric mean of the forward scattervalue squared (FSC²) to obtain an approximation for density of CD47expression on the membrane.

Engraftment of MOLM-13 cells was assessed by using anti-human CD45PE-Cy7 (Pharmingen), anti-mouse CD45.2 APC (clone AL1-4A2), andanti-mouse CD47 Alexa-680 (mIAP301). All samples were resuspended inpropidium iodide containing buffer before analysis to exclude deadcells. FACS data was analyzed using FloJo software (Treestar).

Lentiviral Preparation and Transduction.

pRRL. sin-18.PPT. Tet07.IRES.GFP.pre, CMV, VSV, and tet trans-activator(tTA) plasmids were obtained from Luigi Naldini. The full length murinecDNA for CD47 form 2 was provided by Eric Brown (UCSF). The CD47 cDNAconstruct was ligated into the BamHI/NheI site of Tet-MCS-IRES-GFP.Plasmid DNA was transfected into 293T cells using standard protocols.The supernatant was harvested and concentrated using a Beckman LM-8centrifuge (Beckman). Cells were transduced with Tet orTet-CD47-MCS-IRES-GFP and tTA lentivirus for 48 hours. GFP+ cells weresorted to purity and grown for several generations to ensure stabilityof the transgenes.

Injections.

Cells were injected intravenously into the retro-orbital sinuses ofrecipient mice or via the tail vein as noted. For intra-femoralinjections, cells were injected into the femoral cavity of anesthetizedmice in a volume of 20 □l using a 27-gauge needle. An isofluorane gaschamber was used to anesthetize mice when necessary.

MOLM-13 Cell Engraftment.

Animals were euthanized when moribund and bone marrow, spleen, and liverharvested. Peripheral blood was obtained by tail bleed of the animals 1hour prior to euthanization. Engraftment of MOLM-13 cells in marrow,spleen, and peripheral blood was determined as described above. Tumorburden in the liver was determined by calculating the area of eachvisible tumor nodule using the formula ((length in mm+ width inmm)/2)*π. Area of each nodule was then added together per liver.

Doxycycline administration. Doxycycline hydrochloride (Sigma) was addedto drinking water at a final concentration of 1 mg/mL. Drinking waterwas replaced every 4 days and protected from light. In addition, micereceived a 10 μg bolus of doxycycline by i.p. injection once a week.

Bone Marrow Derived Macrophages (BMDM).

Femurs and tibias were harvested from C57Bl/6 Ka mice and the marrow wasflushed and placed into a sterile suspension of PBS. The bone marrowsuspension was grown in IMDM plus 10% FBS with 10 ng/mL of recombinantmurine macrophage colony stimulating factor (MCSF, Peprotech) for 7-10days.

In Vitro Phagocytosis Assays.

BMDM were harvested by incubation in trypsin/EDTA (Gibco) for 5 minutesand gentle scraping. Macrophages were plated at 5×10⁴ cells per well ina 24-well tissue culture plate (Falcon). After 24 hours, media wasreplaced with serum-free IMDM. After an additional 2 hours, 2.5×10⁵ Tetor Tet-CD47 MOLM-13 cells were added to the macrophage containing wellsand incubated at 37 C.° for the indicated times. After co-incubation,wells were washed thoroughly with IMDM 3 times and examined under anEclipse T5100 (Nikon) using an enhanced green fluorescent protein (GFP)or Texas Red filter set (Nikon). The number of GFP+ or RFP+ cells withinmacrophages was counted and phagocytic index was calculated using theformula: phagocytic index=number of ingested cells/(number ofmacrophages/100). At least 200 macrophages were counted per well. Forflow cytometry analysis of phagocytosis macrophages were harvested afterincubation with MOLM-13 cells using trypsin/EDTA and gentle scraping.Cells were stained with anti-Mac-1 PE antibody and analyzed on a BDFACSAria. Fluorescent and brightfield images were taken separately usingan Eclipse T5100 (Nikon), a super high pressure mercury lamp (Nikon), anendow green fluorescent protein (eGFP) bandpass filter (Nikon) a TexasRed bandpass filter (Nikon), and a RT Slider (Spot Diagnostics) camera.Images were merged with Photoshop software (Adobe).

For in vivo assays, marrow from leg long bones, spleen, and liver wereharvested 2 hours after injecting target cells into RAG2^(−/−),Gc^(−/−)mice. They were prepared into single cell suspensions in PBS plus 2%FCS. Cells were labeled with anti-human CD45 Cy7-PE and anti-mouse F4/80biotin (eBiosciences). Secondary stain was performed withStreptavidin-APC (eBiosciences). Cells that were human CD45−, F4/80+were considered to be macrophages, and GFP+ cells in this fraction wasassessed.

Example 3 Hematopoietic Stem and Progenitor Cells Upregulate CD47 toFacilitate Mobilization and Homing to Hematopoietic Tissues

We show here that hematopoietic stem cells (HSCs) from CD47 deficient(IAP^(−/−)) mice fail to engraft wild-type recipients. As expected,these cells are rapidly cleared by host macrophages, whereas IAP^(+/+)HSCs are not. When stem and progenitor cells are forced to divide andenter circulation using cyclophosphamide/G-CSF or lipopolysaccharide,CD47 is rapidly up-regulated on these cells. We propose that higherlevels of CD47 in stem cells during stress hematopoiesis andmobilization provides added protection against phagocytosis by activatedmacrophages of the reticuloendothelial system. In support of thishypothesis, we show that IAP^(+/−) cells transplanted into wild-typerecipients lose engraftment over time, whereas wild-type donor cells donot. We conclude that phagocytosis is a significant physiologicalmechanism that clears hematopoietic progenitors over time, and that CD47over-expression is required to prevent phagocytic clearance.

HSCs have the ability to migrate to ectopic niches in fetal and adultlife via the blood stream. Furthermore, HSCs can be prodded into thecirculation using a combination of cytotoxic agents and cytokines thatfirst expand HSC numbers in situ. Once in the blood stream, HSCs mustnavigate the vascular beds of the spleen and liver. Macrophages at thesesites function to remove damaged cells and foreign particles from theblood stream. Furthermore, during inflammatory states, macrophagesbecome more phagocytically active. Hence, additional protection againstphagocytosis might be required for newly arriving stem cells at thesesites.

We determined if CD47 expression on bone marrow stem and progenitorcells had a role in regulation of normal hematopoiesis. CD47 expressionhas been shown to be essential for preventing phagocytosis of red bloodcells, T-cells, and whole bone marrow cells in a transplant setting.Thus, we asked if lack of CD47 would prevent HSCs from engrafting afterbeing delivered intravenously. To test this, we employed the CD47knockout mouse (IAP^(−/−)). These mice develop normally and do notdisplay any gross abnormalities. They do, however, die very quicklyafter intraperitoneal bacterial challenge because neutrophils fail tomigrate to the gut quickly. In addition, cells from these mice fail totransplant into wild-type recipients, but they will engraft in IAP−/−recipients.

We first examined stem and progenitor frequencies in IAP^(+/−) andIAP^(−/−) mice. When examining for cells in the stem and myeloidprogenitor compartment, there was no difference between these mice andwild-type mice (FIG. 18a ). We then tested stem cells from these micefor their ability to form colonies in an in vitro assay. We sortedhighly purified Flk2− CD34− KLS stem cells from these mice and platedthem onto methylcellulose in the presence of a standard cytokinecocktail. We examined colony formation at day 7 and found that there wasno major difference between wild-type and IAP−/− stem cells in thenumber and type of colonies formed (FIG. 18b ).

We then asked if bone marrow cells from IAP^(−/−) mice could rescuerecipient mice from the effects of lethal irradiation. Typically, a doseof 2×10⁵ bone marrow cells will rescue 100% of wild-type recipient micein this assay. We found that IAP^(−/−) bone marrow could not rescuethese recipients (FIG. 18c ). However, administration of these cells didprolong lifespan; normally, mice die between day 12 and 15 afterirradiation, but mice that received IAP^(−/−) bone marrow lived about 7to 10 days longer (FIG. 18c ). We do not yet know the reason for theprolongation of lifespan in this case, although we have observed thatmultipotent progenitors and megakaryocyte erythrocyte progenitors canprolong survival after lethal irradiation, and that contribution fromthese cells following transplant of whole bone marrow may havecontributed to the elongation of survival time.

Next, we sorted Flk-2⁻ CD34⁻ KLS stem cells from wild-type and IAP^(−/−)cells and transplanted them into wild-type recipients along with 2×10⁵competitor cells. None of the mice which received IAP^(−/−) HSCs, ateither a dose of 50 or 500 had any engraftment of donor cells,indicating that CD47 was indeed required for the ability of these cellsto transplant (FIG. 18d-e ). We speculated that this was due tophagocytosis of the cells which lacked CD47, as has been shown forerythrocytes and T-cells. To test this, we enriched c-Kit⁺ cells fromthe bone marrow of wild-type and IAP^(−/−) mice and co-incubated themwith bone marrow derived macrophages. IAP^(−/−) stem and progenitorcells were readily phagocytosed in this assay, whereas wild-type cellswere only minimally phagocytosed (FIG. 18f-g ). Interestingly, whenincubated with IAP^(−/−) macrophages, there was significantly lessphagocytosis of IAP^(−/−) cells, confirming that macrophages from thesemice are indeed abnormal in their phagocytic capacity.

Since mobilization of stem and progenitor cells involves several stepsin which they come into contact with macrophages (egress from the marrowsinusoids, entry into the marrow and liver sinusoids, and in the splenicmarginal zone), we asked if CD47 is up-regulated in the marrow of micewhich have been induced to undergo mobilization. The most commonly usedprotocol involves administering the drug cyclophosphamide (Cy), whichkills dividing (mainly myeloid progenitor) cells, followed by treatmentwith granulocyte colony stimulating factor (G-CSF). This involvesadministering cyclophosphamide on the first day, and then giving G-CSFevery day thereafter. By convention, the first day aftercyclophosphamide administration is called day 0. The peak numbers ofstem cells in the bone marrow is achieved on day 2; from days 3-4 theyegress from the bone marrow into the periphery, and their numbers in thespleen and liver reach a peak at day 5; myeloerythroid progenitors arealso mobilized. There is a characteristic rise in the frequency of stemcells and myeloid progenitors during the mobilization response.

Thus, we administered this mobilization protocol to wild-type mice andsacrificed mice on days 2 through 5. We found that there was a notableincrease of CD47 on c-Kit⁺ bone marrow cells on day 2 (FIG. 19a ). Wefound that there was approximately a four-fold increase in the level ofCD47 on stem and progenitor cells on day 2 of mobilization (FIG. 19b ).The increase was seen at all levels of the myeloid progenitor hierarchy,as LT-HSCs as well as GMPs displayed this increase in CD47 expression(FIG. 19b ). By day 5, when egress from the marrow has largely halted,the levels of CD47 had returned to nearly normal levels. In FIG. 19c ,the mean fluorescence intensity of CD47 expression on GMPs is shown ondays 0 to 5 of mobilization. CD47 levels are actually subnormalfollowing myeloablation on day 0, but they quickly rise to a peak on day2. The expression quickly lowers thereafter and the levels by day 5 areequivalent to steady state.

Endotoxins are also thought to contribute to bone marrow mobilization.This may represent a physiological response to infection, where normalmarrow output of immune cells needs to be increased to clear theoffending pathogens. Lipopolysaccharide (LPS) is a cell wall componentof gram-negative bacteria. It binds to the lipid binding protein (LBP)in the serum, which can then form a complex with CD1411 and toll-likereceptor 4 (TLR-4) 12 on monocytes, macrophages, and dendritic cells.This results in activation of macrophages and results in aproinflammatory response. LPS administration has also been shown toincrease the phagocytic capacity of macrophages. This may be due to thefact that LBP-LPS complexes act as opsonins. rusiffe tested if LPSadministration in mice would affect CD47 expression in stem andprogenitor cells. Mirroring the pattern seen in Cy/G inducedmobilization, LPS caused expansion of stem and progenitor cells by 2days post treatment, followed by migration to the spleen and liver (FIG.19d ). On day 2 after LPS administration, stem and progenitor cells inthe marrow had up-regulated CD47 to a similar degree as in Cy/Gmobilization. By day 5, when the inflammatory response has resolved, thelevels of the protein had dropped to steady-state levels (FIG. 19d ).

Since CD47 was consistently up-regulated in the mobilization response,we decided to test the ability of stem and progenitor cells to mobilizefollowing Cy/G. The CD47 knockout mouse has defects in migration ofneutrophils to sites of inflammation 8 and of dendritic cells tosecondary lymphoid organs. The exact role of CD47 in migration of thesecells is unknown, but it may relate to poor integrin association in thecirculation (CD47 binds to several integrins) or lack of interactionwith SIRP□ on endothelial cells. Hence we reasoned that if CD47 wasinvolved in the migration capacity of these cells in the mobilizationresponse, then IAP−/− mice would display reduced numbers of cells in theperipheral organs after Cy/G.

To test this hypothesis we administered Cy/G to both wild-type andknockout mice and sacrificed mice on days 2-5. For each mouse, weanalyzed the number of stem and progenitor cells in marrow, spleen, andliver. We decided to use the crude KLS population as a surrogate forHSCs because numbers of CD34− cells drops considerably in proliferativestates, making accurate calculation of LT-HSC numbers difficult. SinceGMP are the most expanded of all the populations in mobilization, wedecided to analyze their numbers as well. To calculate absoluteprogenitor count, the total cellularity of marrow, spleen, and liver wasestimated by counting the mononuclear cell number in the whole organ byhemocytometer. For bone marrow, leg long bones were assumed to represent15% of the total marrow. This number was then multiplied by thefrequency of the cell population to determine an absolute count.

We found that there was little difference in mobilization of KLS or GMPbetween wild-type and IAP^(−/−) mice (FIG. 19e ). There was a modestdecrease in the ability of IAP^(−/−) mice to move progenitors to thespleen by day 3, but by days 4 and 5 they had restored normal numbers ofcells to the periphery. The marrow and liver compartments looked similarto wild-type mice. Hence, IAP^(−/−) mice do not have a significantmobilization defect.

Heterozygote IAP^(+/−) erythrocytes have roughly the half the amount ofCD47 as wild-type erythrocytes and platelets. There is also a dosedependent increase in the amount of phagocytosis that occurs inimmunoglobulin opsonized IAP^(+/−) erythrocytes and platelets relativeto wild-type. Our observation that CD47 levels increase in states ofstress and mobilization led us to hypothesize that cells that weregenetically hemizygous for CD47 might be more prone to phagocytosis andclearance by macrophages over time. Hence, we asked if IAP^(+/−) stemcells would be disadvantaged relative to wild-type stem cells inlong-term contribution to hematopoiesis.

We first analyzed the levels of CD47 expressed on IAP^(+/+), IAP^(+/−),and IAP^(−/−) stem cells. FACS analysis of CD34⁻ Flk-2⁻ KLS stem cellsrevealed that the MFI of CD47 on heterozygote HSCs was indeed at roughlyhalf the level of wild-type stem cells (FIG. 20a ). We then transplantedthese cells and examined their ability to engraft and producehematopoietic cells in a recipient. We gave congenic wild-type recipientmice 475 Gy, a sublethal dose of irradiation. We then transplanted onecohort of recipients with 2×10⁶ wild-type whole bone marrow cells, andanother with the same dose of IAP^(+/−) bone marrow cells. Such a dosewould be expected to contain roughly 50-100 HSCs. Since granulocytechimerism in the peripheral blood is a good surrogate marker of stemcell fitness, we analyzed cells from the blood of these recipients atperiodic intervals. When wild-type marrow was transplanted intowild-type recipients, granulocyte chimerism was maintained for up to 40weeks. However, when IAP^(+/−) cells were transplanted, 3 out of 5 micelost donor chimerism over time, despite having a successful engraftmentinitially (FIG. 20b ).

We have observed that CD47 is up-regulated on the surface ofhematopoietic cells in the progression of leukemia. We have also foundan analogous increase in the level of CD47 expression when mice werestimulated to mobilize stem and progenitor cells to the periphery usingCy/G, or when they were challenged with LPS. But why is CD47 upregulatedin these states? Various studies have described a dose-dependent effectfor CD47 in the prevention of phagocytosis. IAP^(+/−) erythrocytes andplatelets, which have half the level of CD47 as wild-type cells, arephagocytosed more readily than their normal counterparts. Evidence alsoindicates that the level of CD47 expression on cells correlates wellwith the ability of the cell to engage the SIRP□ inhibitory receptor onmacrophages. Recently Danska et al reported that the ability of NOD-SCIDmice to support transplantation of human hematopoietic cells correlatedwith a mutation in the SIRPalpha receptor in these mice. Here we showthat stem and progenitor cells that express higher levels of CD47 areless likely to be cleared by phagocytosis.

These studies point to a role for CD47 up-regulation in protectinghematopoietic stem cells during states when they are more prone to beingphagocytosed by macrophages, such as post-myeloablation and duringmobilization. Macrophages have the function of removing aged or damagedcells that they encounter; it seems that they can eliminate damaged stemcells as well. Thus, healthy recovering stem cells might up-regulateCD47 during a mobilization response to prevent clearance, whereasdamaged stem cells fail to do so and are cleared. We speculate that thisis a mechanism by which the hematopoietic system self-regulates itselfto ensure that only healthy, undamaged cells are permitted to surviveand proliferate and utilize resources during high stress states. Themobilization of HSC and progenitors into the bloodstream and thence tohematopoietic sites following LPS induced inflammation is veryinteresting; HSC migrate from blood to marrow using integrin α4β1(Wagers and Weissman, Stem Cells 24(4):1087-94, 2006) and the chemokinereceptor CXCR4 (Wright D E et al., J Exp Med 195(9): 1145-54, 2002). Wehave shown previously that integrin α4α1 binds to VCAM1 on hematopoieticstroma (Mikaye K et al. J Exp Med 173(3):599-607, 1991); VCAM1 is alsothe vascular addressin on vessels that inflammatory T cells use torecognize and enter local sites of cell death and inflammation. Inaddition to expressing the integrin associated protein CD47, itinerantHSC express functional integrin α4α1, leading to the speculation thatmigrating hematopoietic stem and progenitors in states of inflammationmay not only re-seed marrow hematopoiesis, but also participate in localinflammation as well.

Materials and Methods

Mice. C57Bl/6 CD45.1 and C57Bl/6 CD45.2 (wild-type) mice were maintainedin our colony. IAP−/− mice were obtained from Eric Brown (University ofCalifornia, San Francisco). These were bred on C57Bl6/J background andcrossed with our wild-type colony.

Screening. IAP+/− were crossed to each other to generate IAP−/− andIAP+/− offspring. Mice were screened by PCR of tail DNA. The followingprimers were used: 3′ Neo-GCATCGCATTGTCTGAGTAGGTGTCATTCTATTC (SEQ ID NO:14); 5′ IAP-TCACCTTGTTGTTCCTGTACTACAAGCA (SEQ ID NO: 15); 3′IAP-TGTCACTTCGCAAGTGTAGTTCC (SEQ ID NO: 16).

Cell staining and sorting. Staining for mouse stem and progenitor cellswas performed using the following monoclonal antibodies: Mac-1, Gr-1,CD3, CD4, CD8, B220, and Ter119 conjugated to Cy5-PE (eBioscience) wereused in the lineage cocktail, c-Kit PE-Cy7 (eBioscience), Sca-1 Alexa680(e13-161-7, produced in our lab), CD34 FITC (eBioscience),CD16/32(FcGRII/III) APC (Pharmingen), and CD135(Flk-2) PE (eBioscience)were used as previously described to stain mouse stem and progenitorsubsets 21 22. Mouse CD47 antibody (clone mIAP301) was assessed usingbiotinylated antibody produced in our lab. Cells were then stained withstreptavidin conjugated Quantum Dot 605 (Chemicon). Samples wereanalyzed using a FACSAria (Beckton Dickinson).

CD34− Flk2− KLS stem cells were double-sorted using a BD FACSAria.Peripheral blood cells were obtained from tail vein bleed and red cellswere eliminated by Dextran T500 (Sigma) precipitation and ACK lysis.Cells were stained with anti-CD45.1 APC, anti-CD45.2 FITC, anti-Ter119PE (Pharmingen), anti-B220 Cy5-PE (eBiosciences), anti-CD3 Cascade Blue,and anti-Mac-1 Cy7-PE (eBiosciences). Granulocytes were Ter119− B220−CD3− Mac-1+ SSC hi. Cells were analyzed using a BD FACSAria.

All samples were resuspended in propidium iodide containing bufferbefore analysis to exclude dead cells. FACS data was analyzed usingFloJo software (Treestar).

In vitro colony forming assay. LT-HSC were directly clone sorted into a96-well plate containing methycellulose media (Methocult 3100) that wasprepared as described. The media was also supplemented with recombinantmouse stem cell factor (SCF), interleukin (IL)-3, IL-11,granulocyte-macrophage colony stimulating factor (GM-CSF),thrombopoietin (Tpo) and erythropoietin (Epo). Colonies were scored forCFU-G, CFU-M, CFU-GM, CFU-GEMM, and Meg.

Cell transfers. For whole bone marrow transfers, IAP+/+, IAP+/−, orIAP−/− cells were freshly isolated from leg long bones. Cells werecounted using a hemacytometer and resuspended in PBS+2% FCS at 100 uL.For some experiments, CD45.1 cells from C57Bl/6 Ka CD45.1 mice were usedas donors into CD45.2 wild-type mice.

For sorted cells, cells were sorted into PBS buffer at the correct dose(i.e. 50 or 500 cells per tube) and resuspended in 100 uL of PBS+2% FCS.For competition experiments, 2×10⁵ freshly isolated whole bone marrowcells from C57Bl/6 CD45.1 were added to the 100 uL stem cell suspension.

C57Bl/6 Ka CD45.1 or C57Bl/6 J CD45.2 recipient mice were irradiatedusing a cesium source at the doses indicated. Sub-lethal dose was 4.75Gray and lethal dose was a split dose of 9.5 Gray. Cells weretransferred using a 27-gauge syringe into the retro-orbital sinuses ofmice anesthetized with isofluorane.

Mobilization assay. Mice were mobilized with cyclophosphamide (Sigma)(200 mg/kg) and G-CSF (Neupogen) (250 □g/kg) as previously described.Bacterial LPS from E. coli 055:B5 (Sigma) was administered at a dose of40 mg/kg into the peritoneal cavity.

For analysis of mobilized organs, whole spleen, whole liver, and leglong bones were prepared in a single cell suspension. Cell density wasdetermined using a hemacytometer to determine overall cellularity ofhematopoietic cells in these organs.

Enrichment of c-Kit⁺ cells. Whole mouse marrow was stained with CD117microbeads (Miltenyi). c-Kit⁺ cells were selected on an AutoMACS Midicolumn (Miltenyi) using a magnetic separator.

In vitro phagocytosis assay. BMDM were prepared as previously described.c-Kit enriched bone marrow cells were stained with CFSE (Invitrogen)prior to the assay. 2.5×10⁵ c-Kit enriched cells were plated with 5×10⁴macrophages. Macrophages and c-Kit cells were obtained from eitherIAP^(+/+) or IAP^(−/−) mice. Cells were incubated for 2 hours andphagocytic index was determined. Photographs were taken as describedpreviously.

Example 4 CD47 is an Independent Prognostic Factor and TherapeuticAntibody Target on Human Acute Myeloid Leukemia Cells

Acute myelogenous leukemia (AML) is organized as a cellular hierarchyinitiated and maintained by a subset of self-renewing leukemia stemcells (LSC). We hypothesized that increased CD47 expression on AML LSCcontributes to pathogenesis by inhibiting their phagocytosis through theinteraction of CD47 with an inhibitory receptor on phagocytes. We foundthat CD47 was more highly expressed on AML LSC than their normalcounterparts, and that increased CD47 expression predicted worse overallsurvival in 3 independent cohorts of adult AML patients. Furthermore,blocking monoclonal antibodies against CD47 preferentially enabledphagocytosis of AML LSC by macrophages in vitro, and inhibited theirengraftment in vivo. Finally, treatment of human AML-engrafted mice withanti-CD47 antibody eliminated AML in vivo. In summary, increased CD47expression is an independent poor prognostic factor that can be targetedon human AML stem cells with monoclonal antibodies capable ofstimulating phagocytosis of LSC.

Results

CD47 is More Highly Expressed on AML LSC than their Normal Counterpartsand is Associated with the FLT3-ITD Mutation.

In our investigation of several mouse models of myeloid leukemia, weidentified increased expression of CD47 on mouse leukemia cells comparedto normal bone marrow. This prompted investigation of CD47 expression onhuman AML LSC and their normal counterparts. Using flow cytometry, CD47was more highly expressed on multiple specimens of AML LSC than normalbone marrow HSC and MPP (FIG. 6). This increased expression extended tothe bulk leukemia cells, which expressed CD47 similarly to theLSC-enriched fraction.

Examination of a subset of these samples indicated that CD47 surfaceexpression correlated with CD47 mRNA expression. To investigate CD47expression across morphologic, cytogenetic, and molecular subgroups ofAML, gene expression data from a previously described cohort of 285adult patients were analyzed (Valk et al., 2004 N Engl J Med 350,1617-1628). No significant difference in CD47 expression among FAB(French-American-British) subtypes was found. In most cytogeneticsubgroups, CD47 was expressed at similar levels, except for casesharboring t(8;21)(q22;q22), a favorable risk group which had astatistically significant lower CD47 expression. In molecularlycharacterized AML subgroups, no significant association was foundbetween CD47 expression and mutations in the tyrosine kinase domain ofFLT3 (FLT3-TKD), over-expression of EVI1, or mutations in CEBPA, NRAS,or KRAS. However, higher CD47 expression was strongly correlated withthe presence of FLT3-ITD (p<0.001), which is observed in nearly onethird of AML, with normal karyotypes and is associated with worseoverall survival. This finding was separately confirmed in twoindependent datasets of 214 and 137 AML patients (Table 1).

TABLE 1 Clinical and Molecular Characteristics of AML Samples from theValidation Cohort and Comparison Between Low CD47 and High CD47Expression Groups Overall Low CD47 High CD47 Clinical Feature* n = 137 n= 95 n = 37 P† Age, yrs. 0.26 Median 46 47 46 Range 10-60 24-60 16-60WBC, ×10⁹/L <0.01 Median 24 17 35 Range  1-238  1-178  1-238 Centrallyreviewed FAB 0.29 Classification, no. (%) M0 11 (8)  9 (9) 2 (5) M1 28(20) 16 (17)  2 (32) M2 36 (26) 22 (23) 11 (30) M4 33 (24) 25 (26)  8(22) M5 19 (14) 16 (17) 3 (8) M6 2 (1) 2 (1) 0 (0) Unclassified 6 (4) 4(4) 0 (0) FLT3-ITD, no. (%) <0.05 Negative 84 (61) 63 (66) 17 (46)Positive 53 (39) 32 (34) 20 (54) FLT3-TKD, no. (%) 0.24 Negative 109(87)  78 (89) 27 (79) Positive 17 (13) 10 (11)  7 (21) NPM1, no. (%)0.10 Wild-Type 55 (45) 41 (49) 10 (30) Mutated 66 (55) 43 (51) 23 (70)CEBPA, no. (%) 1 Wild-Type 100 (86)  70 (86) 27 (87) Mutated 16 (14) 11(14)  4 (13) MLL-PTD, no. (%) 1 Negative 121 (93)  83 (92) 34 (94)Positive 9 (7) 7 (8) 2 (6) Event-free survival 0.004 Median, mos. 14  17.1   6.3 Disease-free at 3 yrs, % (95% CI) 36 (27-44) 41 (31-52)  22 (8-36) Overall survival 0.002 Median, mos.   18.5   22.1   9.1Alive at 3 yrs, % (95% CI)   39 (31-48)   44 (33-55)   26 (12-41)Complete remission rate, no. (%) CR after 1st Induction, no. (%)   60(46%)   46 (48%)   14 (35%) 0.33 CR after 2nd Induction, no. (%)   84(74%)   64 (75%)   20 (69%) 0.63 Randomization to 2ndary consolidativetherapy Allogeneic-HSCT, no. (%)   29 (22%)   25 (26%)   4 (11%) 0.09Autologous-HSCT, no. (%)   23 (17%)   17 (18%)   6 (16%) 0.98 *Tabulatedclinical and molecular characteristics at diagnosis. WBC indicates whiteblood cell conar; FAB, French-American-British; FLT3-ITD, internaltandem duplication of the FLT3 gene (for 10 cases with missing FLT3-ITDstatus, the predicted FLT3-ITD status based on gene expression wassubstituted using method of Ballinger et al, 2005); FLT3-TKD, tyrosinekinase domain mutation of the FLT3 gene; NPMI, mutation of the NPMIgene; MLL-PTD, partial tandem duplication of the MLL gene; and CEBPA,mutation of the CEBPA gene. CR, complete remission. CR was assessed bothafter first and second induction regimens, which comprised ICE (

, etoposide, cytarabine) or A-HAM (all-trans retinoic acid and high-dosecytrabine plus mitoxantrone). Autologous-HSCT: autologoustransplantation; Allogeneic-HSCT, allogenic transplantation. †P valuecompares differences in molecular and clinical characteristics atdiagnosis between patients with low and high CD47 mRNA expressionvalues. CD47 expression was dichromized based on an optimal cut pointfor overall survival stranfication that we identified on an independentmicroarry dataset published (Valk et al, 2004) as described tosupplemental methods.

indicates data missing or illegible when filed

Identification and Separation of Normal and Leukemic Progenitors fromthe Same Patient Based on Differential CD47 Expression.

In the LSC-enriched Lin−CD34+CD38− fraction of specimen SU008, a rarepopulation of CD47lo-expressing cells was detected, in addition to themajority CD47^(hi)-expressing cells (FIG. 21A). These populations wereisolated by fluorescence-activated cell sorting (FACS) to >98% purityand either transplanted into newborn NOG mice or plated into completemethylcellulose. The CD47^(hi) cells failed to engraft in vivo or formany colonies in vitro, as can be observed with some AML specimens.

However, the CD47^(lo) cells engrafted with normal myelo-lymphoidhematopoiesis in vivo and formed numerous morphologically normal myeloidcolonies in vitro (FIG. 21B,C). This specimen harbored the FLT3-ITDmutation, which was detected in the bulk leukemia cells (FIG. 21D). Thepurified CD47^(hi) cells contained the FLT3-ITD mutation, and therefore,were part of the leukemic clone, while the CD47^(lo) cells did not.Human cells isolated from mice engrafted with the CD47^(lo) cellscontained only wild type FLT3, indicating that the CD47^(lo) cellscontained normal hematopoietic progenitors.

Increased CD47 Expression in Human AML is Associated with Poor ClinicalOutcomes.

We hypothesized that increased CD47 expression on human AML contributesto pathogenesis. From this hypothesis, we predicted that AML with higherexpression of CD47 would be associated with worse clinical outcomes.Consistent with this hypothesis, analysis of a previously describedgroup of 285 adult AML patients with diverse cytogenetic and molecularabnormalities (Valk et al., 2004) revealed that a dichotomousstratification of patients into low CD47 and high CD47 expression groupswas associated with a significantly increased risk of death in the highexpressing group (p=0.03). The association of overall survival with thisdichotomous stratification of CD47 expression was validated in a secondtest cohort of 242 adult patients (Metzeler et al., 2008 Blood) withnormal karyotypes (NK-AML) (p=0.01).

Applying this stratification to a distinct validation cohort of 137adult patients with normal karyotypes (Bullinger et al., 2008 Blood 111,4490-4495), we confirmed the prognostic value of CD47 expression forboth overall and event-free survival (FIG. 22). Analysis of clinicalcharacteristics of the low and high CD47 expression groups in thiscross-validation cohort also identified statistically significantdifferences in white blood cell (WBC) count and FLT3-ITD status, and nodifferences in rates of complete remission and type of consolidativetherapy including allogeneic transplantation (Table 1). Kaplan-Meieranalysis demonstrated that high CD47 expression at diagnosis wassignificantly associated with worse event-free and overall survival(FIG. 22 A,B). Patients in the low CD47 expression group had a medianevent-free survival of 17.1 months compared to 6.8 months in the highCD47 expression group, corresponding to a hazard ratio of 1.94 (95%confidence interval 1.30 to 3.77, p=0.004). For overall survival,patients in the low CD47 expression group had a median of 22.1 monthscompared to 9.1 months in the high CD47 expression group, correspondingto a hazard ratio of 2.02 (95% confidence interval 1.37 to 4.03,p=0.002). When CD47 expression was considered as a continuous variable,increased expression was also associated with a worse event-free(p=0.02) and overall survival (p=0.02).

Despite the association with FLT3-ITD (Table 1), increased CD47expression at diagnosis was significantly associated with worseevent-free and overall survival in the subgroup of 74 patients withoutFLT3-ITD, when considered either as a binary classification (FIG. 22C,D)or as a continuous variable (p=0.02 for both event-free and overallsurvival). In multivariable analysis considering age, FLT3-ITD status,and CD47 expression as a continuous variable, increased CD47 expressionremained associated with worse event-free survival with a hazard ratioof 1.33 (95% confidence interval 1.03 to 1.73, p=0.03) and overallsurvival with a hazard ratio of 1.31 (95% confidence interval 1.00 to1.71, p=0.05) (Table 2).

TABLE 2 Outcome Measure/ Variables Considered HR 95% CI P Event-freesurvival CD47 expression, continuous, 1.33 1.03-1.73 0.03 per 2-foldincrease FLT3-ITD, positive vs. negative 2.21 1.39-3.53 <0.001 Age, peryear 1.03 1.00-1.06 0.03 Overall survival CD47 expression, continuous,1.31 1.00-1.71 0.05 per 2-fold increase FLT3-ITD, positive vs. negative2.29 1.42-3.68 <0.001 Age, per year 1.03 1.01-1.06 0.01

Monoclonal Antibodies Directed Against Human CD47 Preferentially EnablePhagocytosis of AML LSC by Human Macrophages.

We hypothesized that increased CD47 expression on human AML contributesto pathogenesis by inhibiting phagocytosis of leukemia cells, leading usto predict that disruption of the CD47-SIRP□ interaction with amonoclonal antibody directed against CD47 will preferentially enable thephagocytosis of AML LSC. Several anti-human CD47 monoclonal antibodieshave been generated including some capable of blocking the CD47-SIRP□interaction (B6H12.2 and BRIC126) and others unable to do so (2D3)(Subramanian et al., 2006 Blood 107, 2548-2556). The ability of theseantibodies to enable phagocytosis of AML LSC, or normal human bonemarrow CD34+ cells, by human macrophages in vitro was tested. Incubationof AML LSC with human macrophages in the presence of IgG1 isotypecontrol antibody or mouse anti-human CD45 IgG1 monoclonal antibody didnot result in significant phagocytosis as determined by eitherimmunofluorescence microscopy (FIG. 8A) or flow cytometry. However,addition of the blocking anti-CD47 antibodies B6H12.2 and BRIC126, butnot the non-blocking 2D3, enabled phagocytosis of AML LSC (FIG. 8A,C).No phagocytosis of normal CD34+ cells was observed with any of theantibodies (FIG. 8C).

Monoclonal Antibodies Directed Against Human CD47 Enable Phagocytosis ofAML LSC by Mouse Macrophages.

The CD47-SIRP□ interaction has been implicated as a critical regulatorof xenotransplantation rejection in several cross species transplants;however, there are conflicting reports of the ability of CD47 from onespecies to bind and stimulate SIRP□ of a different species. In order todirectly assess the effect of inhibiting the interaction of human CD47with mouse SIRP□, the in vitro phagocytosis assays described above wereconducted with mouse macrophages. Incubation of AML LSC with mousemacrophages in the presence of IgG1 isotype control antibody or mouseanti-human CD45 IgG1 monoclonal antibody did not result in significantphagocytosis as determined by either immunofluorescence microscopy (FIG.8B) or flow cytometry. However, addition of the blocking anti-CD47antibodies B6H12.2 and BRIC126, but not the non-blocking 2D3, enabledphagocytosis of AML LSC (FIG. 8B,C).

A Monoclonal Antibody Directed Against Human CD47 Inhibits AML LSCEngraftment and Eliminates AML In Vivo.

The ability of the blocking anti-CD47 antibody B6H12.2 to target AML LSCin vivo was tested. First, a pre-coating strategy was utilized in whichAML LSC were purified by FACS and incubated with IgG1 isotype control,anti-human CD45, or anti-human CD47 antibody. An aliquot of the cellswas analyzed for coating by staining with a secondary antibodydemonstrating that both anti-CD45 and anti-CD47 antibody bound the cells(FIG. 10A). The remaining cells were transplanted into newborn NOG micethat were analyzed for leukemic engraftment 13 weeks later (FIG. 10B).In all but one mouse, the isotype control and anti-CD45 antibody coatedcells exhibited long-term leukemic engraftment. However, most micetransplanted with cells coated with anti-CD47 antibody had no detectableleukemia engraftment.

Next, a treatment strategy was utilized in which mice were firstengrafted with human AML LSC and then administered daily intraperitonealinjections of 100 micrograms of either mouse IgG or anti-CD47 antibodyfor 14 days, with leukemic engraftment determined pre- andpost-treatment. Analysis of the peripheral blood showed near completeelimination of circulating leukemia in mice treated with anti-CD47antibody, often after a single dose, with no response in control mice(FIG. 23A,B). Similarly, there was a significant reduction in leukemicengraftment in the bone marrow of mice treated with anti-CD47 antibody,while leukemic involvement increased in control IgG-treated mice (FIG.23 C,D). Histologic analysis of the bone marrow identified monomorphicleukemic blasts in control IgG-treated mice (FIG. 23E, panels 1,2) andcleared hypocellular areas in anti-CD47 antibody-treated mice (FIG. 23E,panels 4,5). In the bone marrow of some anti-CD47 antibody-treated micethat contained residual leukemia, macrophages were detected containingphagocytosed pyknotic cells, capturing the elimination of human leukemia(FIG. 23E, panels 3,6).

We report here the identification of higher expression of CD47 on AMLLSC compared to their normal counterparts and hypothesize that increasedexpression of CD47 on human AML contributes to pathogenesis byinhibiting phagocytosis of these cells through the interaction of CD47with SIRP□. Consistent with this hypothesis, we demonstrate thatincreased expression of CD47 in human AML is associated with decreasedoverall survival. We also demonstrate that disruption of the CD47-SIRP□interaction with monoclonal antibodies directed against CD47preferentially enables phagocytosis of AML LSC by macrophages in vitro,inhibits the engraftment of AML LSC, and eliminates AML in vivo.Together, these results establish the rationale for considering the useof an anti-CD47 monoclonal antibody as a novel therapy for human AML.

The pathogenic influence of CD47 appears mechanistically distinct fromthe two main complementing classes of mutations in a model proposed forAML pathogenesis. According to this model, class I mutations, whichprimarily impact proliferation and apoptosis (for example, FLT3 andNRAS), and class II mutations, which primarily impair hematopoietic celldifferentiation (for example, CEBPA, MLL, and NPM1), cooperate inleukemogenesis. As demonstrated here, CD47 contributes to pathogenesisvia a distinct mechanism, conferring a survival advantage to LSC andprogeny blasts through evasion of phagocytosis by the innate immunesystem. While strategies for the evasion of immune responses have beendescribed for many human tumors, we believe that increased CD47expression represents the first such immune evasion mechanism withprognostic and therapeutic implications for human AML.

Higher CD47 Expression is a Marker of Leukemia Stem Cells and Prognosticfor Overall Survival in AML. AML LSC are enriched in the Lin−CD34+CD38−fraction, which in normal bone marrow contains HSC and MPP. Theidentification of cell surface molecules that can distinguish betweenleukemic and normal stem cells is essential for flow cytometry-basedassessment of minimal residual disease (MRD) and for the development ofprospective separation strategies for use in cellular therapies. Severalcandidate molecules have recently been identified, including CD123,CD96, CLL-1, and now CD47. CD123 was the first molecule demonstrated tobe more highly expressed on AML LSC compared to normal HSC-enrichedpopulations. We previously identified AML LSC-specific expression ofCD96 compared to normal HSC, and demonstrated that only CD96+, and notCD96−, leukemia cells were able to engraft in vivo.

CLL-1 was identified as an AML LSC-specific surface molecule expressedon most AML samples and not normal HSC; importantly, the presence ofLin−CD34+CD38−CLL-1+ cells in the marrow of several patients inhematologic remission was predictive of relapse. Here we demonstratethat not only is CD47 more highly expressed on AML LSC compared tonormal HSC and MPP, but that this differential expression can be used toseparate normal HSC/MPP from LSC. This is the first demonstration of theprospective separation of normal from leukemic stem cells in the samepatient sample, and offers the possibility of LSC-depleted autologousHSC transplantation therapies.

We initially identified higher expression of CD47 on AML LSC, but notedthat expression in bulk blasts was the same. Because of this, we decidedto utilize published gene expression data on bulk AML to investigate therelationship between CD47 expression and clinical outcomes. Consistentwith our hypothesis, we found that increased CD47 expression wasindependently predictive of a worse clinical outcome in AML patientswith a normal karyotype, including the subset without the FLT3-ITDmutation, which is the largest subgroup of AML patients. As thisanalysis was dependent on the relative expression of CD47 mRNA, aquantitative PCR assay for AML prognosis may be based on the level ofCD47 expression. Such an assay could be utilized in risk adaptedtherapeutic decision making, particularly in the large subgroup of AMLpatients with normal karyotypes who lack the FLT3-ITD mutation.

Targeting of CD47 on AML LSC with Therapeutic Monoclonal Antibodies Cellsurface molecules preferentially expressed on AML LSC compared to theirnormal counterparts are candidates for targeting with therapeuticmonoclonal antibodies. Thus far, several molecules have been targeted onAML including CD33, CD44, CD123, and now CD47. CD33 is the target of themonoclonal antibody conjugate gemtuzumab ozogamicin (Mylotarg), which isapproved for the treatment of relapsed AML in older patients. Targetingof CD44 with a monoclonal antibody was shown to markedly reduce AMLengraftment in mice, with evidence that it acts specifically on LSC toinduce differentiation. A monoclonal antibody directed against CD123 wasrecently reported to have efficacy in reducing AML LSC function in vivo.Here we report that a monoclonal antibody directed against CD47 is ableto stimulate phagocytosis of AML LSC in vitro and inhibit engraftment invivo.

Several lines of evidence suggest that targeting of CD47 with amonoclonal antibody likely acts by disrupting the CD47-SIRP□interaction, thereby preventing a phagocytic inhibitory signal. First,two blocking anti-CD47 antibodies enabled AML LSC phagocytosis, whileone non-blocking antibody did not, even though all three bind the cellssimilarly. Second, in the case of the B6H12.2 antibody used for most ofour experiments, the isotype-matched anti-CD45 antibody, which alsobinds LSC, failed to produce the same effects. In fact, the B6H12.2antibody is mouse isotype IgG1, which is less effective at engagingmouse Fc receptors than antibodies of isotype IgG2a or IgG2b.

For human clinical therapies, blocking CD47 on AML LSC with humanizedmonoclonal antibodies promotes LSC phagocytosis through a similarmechanism, as indicated by the human macrophage-mediated in vitrophagocytosis (FIG. 8A,C). Higher CD47 expression is detected on AML LSC;however, CD47 is expressed on normal tissues, including bone marrow HSC.We identified a preferential effect of anti-CD47 antibodies in enablingthe phagocytosis of AML LSC compared to normal bone marrow CD34+ cellsby human macrophages in vitro. In fact, no increased phagocytosis ofnormal CD34+ cells compared to isotype control was detected,demonstrating that blocking CD47 with monoclonal antibodies is a viabletherapeutic strategy for human AML.

The experimental evidence presented here provides the rationale foranti-CD47 monoclonal antibodies as monotherapy for AML. However, suchantibodies may be equally, if not more effective as part of acombination strategy. The combination of an anti-CD47 antibody, able toblock a strong inhibitory signal for phagocytosis, with a secondantibody able to bind a LSC-specific molecule (for example CD96) andengage Fc receptors on phagocytes, thereby delivering a strong positivesignal for phagocytosis, may result in a synergistic stimulus forphagocytosis and specific elimination of AML LSC. Furthermore,combinations of monoclonal antibodies to AML LSC that include blockinganti-CD47 and human IgG1 antibodies directed against two other cellsurface antigens will be more likely to eliminate leukemia cells withpre-existing epitope variants or antigen loss that are likely to recurin patients treated with a single antibody.

Experimental Procedures

Human Samples.

Normal human bone marrow mononuclear cells were purchased from AllCellsInc. (Emeryville, Calif.). Human acute myeloid leukemia samples (FIG.1A) were obtained from patients at the Stanford University MedicalCenter with informed consent, according to an IRB-approved protocol(Stanford IRB#76935 and 6453). Human CD34-positive cells were enrichedwith magnetic beads (Miltenyi Biotech).

Flow Cytometry Analysis and Cell Sorting.

A panel of antibodies was used for analysis and sorting of AML LSC(Lin−CD34+CD38−CD90−, where lineage included CD3, CD19, and CD20), HSC(Lin−CD34+CD38−CD90+), and MPP (Lin−CD34+CD38−CD90−CD45RA−) aspreviously described (Majeti et al., 2007). Analysis of CD47 expressionwas performed with an anti-human CD47 PE antibody (clone B6H12, BDBiosciences, San Jose Calif.).

Genomic DNA Preparation and Analysis of FLT3-ITD by PCR. Genomic DNA wasisolated from cell pellets using the Gentra Puregene Kit according tothe manufacturer's protocol (Gentra Systems, Minneapolis, Minn.).FLT3-ITD status was screened by PCR using primers that generated awild-type product of 329 bp and ITD products of variable larger sizes.

Anti-Human CD47 Antibodies.

Monoclonal mouse anti-human CD47 antibodies included: BRIC126, IgG2b(Abcam, Cambridge, Mass.), 2D3, IgG1 (Ebiosciences. San Diego, Calif.),and B6H12.2, IgG1. The B6H12.2 hybridoma was obtained from the AmericanType Culture Collection (Rockville, Md.). Antibody was either purifiedfrom hybridoma supernatant using protein G affinity chromatographyaccording to standard procedures or obtained from BioXCell (Lebanon,N.H.).

Methylcellulose Colony Assay.

Methylcellulose colony formation was assayed by plating sorted cellsinto a 6-well plate, each well containing 1 ml of completemethylcellulose (Methocult GF+ H4435, Stem Cell Technologies). Plateswere incubated for 14 days at 37° C., then scored based on morphology.

In Vitro Phagocytosis Assays.

Human AML LSC or normal bone marrow CD34+ cells were CF SE-labeled andincubated with either mouse or human macrophages in the presence of 7μg/ml IgG1 isotype control, anti-CD45 IgG1, or anti-CD47 (clonesB6H12.2, BRIC126, or 2D3) antibody for 2 hours. Cells were then analyzedby fluorescence microscopy to determine the phagocytic index (number ofcells ingested per 100 macrophages). In some cases, cells were thenharvested and stained with either a mouse or human macrophage marker andphagocytosed cells were identified by flow cytometry asmacrophage+CFSE+. Statistical analysis using Student's t-test wasperformed with GraphPad Prism (San Diego, Calif.).

In Vivo Pre-Coating Engraftment Assay. LSC isolated from AML specimenswere incubated with 28 ug/mL of IgG1 isotype control, anti-CD45 IgG1, oranti-CD47 IgG1 (B6H12.2) antibody at 4° C. for 30 minutes. A smallaliquot of cells was then stained with donkey anti-mouse PE secondaryantibody (Ebioscience) and analyzed by flow cytometry to assess coating.Approximately 10⁵ coated LSC were then transplanted into each irradiatednewborn NOD.Cg-PrkdcscidIl2rgtmlWjl/SzJ (NOG) mouse. Mice weresacrificed 13 weeks post-transplantation and bone marrow was analyzedfor human leukemia engraftment (hCD45+hCD33+) by flow cytometry (Majetiet al., 2007 Cell Stem Cell 1, 635-645). The presence of human leukemiawas confirmed by Wright-Giemsa staining of hCD45+ cells and FLT3-ITDPCR. Statistical analysis using Student's t-test was performed withGraphPad Prism (San Diego, Calif.).

In Vivo Antibody Treatment of AML Engrafted Mice.

1-25×10⁵ FACS-purified LSC were transplanted into NOG pups. Eight totwelve weeks later, human AML engraftment (hCD45+CD33+ cells) wasassessed in the peripheral blood and bone marrow by tail bleed andaspiration of the femur, respectively. Engrafted mice were then treatedwith daily intraperitoneal injections of 100 micrograms of anti-CD47antibody or IgG control for 14 days. On day 15 mice were sacrificed andthe peripheral blood and bone marrow were analyzed for AML.

AML Patients, Microarray Gene Expression Data, and Statistical Analysis.

Gene expression and clinical data were analyzed for three previouslydescribed cohorts of adult AML patients: (1) a training dataset of 285patients with diverse cytogenetic and molecular abnormalities describedby Valk et al., (2) a test dataset of 242 patients with normalkaryotypes described by Metzeler et al., and (3) a validation dataset of137 patients with normal karyotypes described by Bullinger et al. Theclinical end points analyzed included overall and event-free survival,with events defined as the interval between study enrollment and removalfrom the study owing to a lack of complete remission, relapse, or deathfrom any cause, with data censored for patients who did not have anevent at the last follow-up visit.

FLT3-ITD PCR.

All reactions were performed in a volume of 50 □l containing 5 □l of10×PCR buffer (50 mM KCL/10 nM Tris/2 mM MgCl2/0.01% gelatin), 1 □l of10 mM dNTPs, 2 units of Taq polymerase (Invitrogen), 1 ul of 10□Mforward primer 11F (5′-GCAATTTAGGTATGAAAGCCAGC-3′; SEQ ID NO: 17) andreverse primer 12R (5′-CTTTCAGCATTTTGACGGCAACC-3′; SEQ ID NO: 18), and10-50 ng of genomic DNA. PCR conditions for amplification of the FLT3gene were 40 cycles of denaturation (30 sec at 95° C.) annealing (30 secat 62° C.), and extension (30 sec at 72° C.).

Preparation of Mouse and Human Macrophages.

BALB/c mouse bone marrow mononuclear cells were harvested and grown inIMDM containing 10% FBS supplemented with 10 ng/mL recombinant murinemacrophage colony stimulating factor (M-CSF, Peprotech, Rocky Hill,N.J.) for 7-10 days to allow terminal differentiation of monocytes tomacrophages. Human peripheral blood mononuclear cells were prepared fromdiscarded normal blood from the Stanford University Medical Center.Monocytes were isolated by adhering mononuclear cells to culture platesfor one hour at 37° C., after which non-adherent cells were removed bywashing. The remaining cells were >95% CD14 and CD11b positive. Adherentcells were then incubated in IMDM plus 10% human serum (ValleyBiomedical, Winchester, Va.) for 7-10 days to allow terminaldifferentiation of monocytes to macrophages.

In Vitro Phagocytosis Assay.

BMDM or peripheral blood macrophages were harvested by incubation intrypsin/EDTA (Gibco/Invitrogen) for 5 minutes followed by gentlescraping. 5×10⁴ macrophages were plated in each well of a 24-well tissueculture plate in 10% IMDM containing 10% FBS. After 24 hours, media wasreplaced with serum-free IMDM and cells were cultured an additional 2hours. LSC were fluorescently labeled with CFSE according to themanufacturer's protocol (Invitrogen). 2×10⁴ CFSE-labeled LSC were addedto the macrophage-containing wells along with 7 □g/mL of IgG1 isotype(Ebiosciences), anti-CD45 (clone HI30, Ebiosciences), or anti-CD47antibody, and incubated for 2 hours. Wells were then washed 3 times withIMDM and examined under an Eclipse T5100 immunofluorescent microscope(Nikon) using an enhanced green fluorescent protein filter able todetected CFSE fluorescence. The number of CFSE positive cells withinmacrophages was counted and the phagocytic index was determined as thenumber of ingested cells per 100 macrophages. At least 200 macrophageswere counted per well. Flourescent and brightfield images were takenseparately and merged with Image Pro Plus (Media Cybernetics, Bethesda,Md.). In FIG. 22A,B, the three left images are presented at 200×magnification, with the anti-CD47 right image at 400× magnification. Forflow cytometry analysis of phagocytosis, the cells were then harvestedfrom each well using trypsin/EDTA. Cell suspensions were then stainedwith a mouse macrophage antibody anti-mouse F4/80-PECy7 (Ebiosciences)or anti-human CD14-PECy7 (Ebiosciences) and analyzed on a FACSAria.Phagocytosed LSC were defined as either CFSE+F4/80+ or CFSE+CD14+ cellswhen incubated with murine or human macrophages, respectively.

Microarray Gene Expression Data.

Panel A of Supplemental FIG. 22 describes the main microarray datasetsanalyzed herein, including the training, test, and validation cohorts.Training Set: Gene expression data, cytogenetics data, and moleculardata for the 285 and 465 patients with AML profiled with AffymetrixHG-U133A and HG-U133 Plus 2.0 microarrays by Valk et al. andJongen-Lavrencic et al. respectively, were obtained from the GeneExpression Omnibus using the corresponding accession numbers (GSE1159and GSE6891). Outcome data were only available for the former dataset,and the corresponding clinical information were kindly provided by theauthors. This cohort is presented as the “training” dataset. The latterdataset was used to confirm univariate associations with karyotype andmolecular mutations described in the former. However, these two datasetsoverlapped in that 247 of the 285 patients in the first study wereincluded in the second, and were accordingly excluded in validation ofthe association of FLT3-ITD with CD47 expression in the 2nd dataset.Using NetAffx4, RefSeq5, and the UCSC Genome Browser6, we identified211075_s_at and 213857_s_at as Affymetrix probe sets on the U133 Plus2.0 microarray mapping exclusively to constitutively transcribed exonsof CD47. The geometric mean of the base-2 logarithms of these two probesets was employed in estimating the mRNA expression level for CD47, andcorresponding statistical measures for associations with FABclassification, karyotype, and molecular mutations. Because the dataprovided by Valk et al. as GSE1159 were Affymetrix intensitymeasurements, we converted these intensities to normalized base-2logarithms of ratios to allow comparison to the correspondingmeasurements from cDNA microarrays using a conventional scheme.Specifically, we first (1) normalized raw data using CEL files from all291 microarrays within this dataset using gcRMA8, then (2) generatedratios by dividing the intensity measurement for each gene on a givenarray by the average intensity of the gene across all arrays, (3)log-transformed (base 2) the resulting ratios, and (4) median centeredthe expression data across arrays then across genes. For the assessmentof the prognostic value of CD47, we employed the probe set 213857_s_atfrom the Affymetrix HG-U133A and HG-U133 Plus 2.0 microarrays, given itssimilar expression distribution (Supplemental FIG. 3B), and consideringits position within the mRNA transcript as compared with cDNA clones onthe Stanford cDNA microarrays as annotated within the NetAffx resource.

Test Set: Gene expression and clinical data for the 242 adult patientswith NKAML profiled with Affymetrix HG-U133A and HG-U133 Plus 2.0microarrays by Metzeler et al. were obtained from the Gene ExpressionOmnibus using the corresponding accession numbers (GSE12417). Since rawdata were not available for this dataset, for purposes of assessing theprognostic value of CD47, we employed the normalized datasets providedby the authors (base 2 logarithms) and assessed expression of CD47 usingthe probe set 213857_s_at on the corresponding microarrays.

Validation Set: Gene expression data for the 137 patients with normalkaryotype AML profiled with cDNA microarrays by Bullinger et al. wereobtained from the Stanford Microarray Database10. The correspondingclinical information including outcome data and FLT3 mutation statuswere kindly provided by the authors. Using the original annotations ofmicroarray features as well as SOURCE11, RefSeq5, and the UCSC GenomeBrowser6, we identified IMAGE:811819 as a sequence verified cDNA clonemapping to the constitutively transcribed 3′ terminal exon of CD47 onthe corresponding cDNA microarrays.

Details of Treatment: AML patients described by Valk et al. (trainingset), were treated according to several protocols of the Dutch-BelgianHematology-Oncology Cooperative group. The majority (90%) of the NK-AMLpatients described by Metzeler et al. (test set) were treated perprotocol AMLCG-1999 of the German AML Cooperative Group, with allpatients receiving intensive double-induction and consolidationchemotherapy. All 137 NK-AML patients described by Bullinger et al.(validation set) received standard-of-care intensified treatmentregimens (protocol AML HD98A), which included 2 courses of inductiontherapy with idarubicin, cytarabine, and etoposide, one consolidationcycle of high-dose cytarabine and mitoxantrone (HAM), followed by randomassignment to a late consolidation cycle of HAM versus autologoushematopoietic cell transplantation in case no HLA identical family donorwas available for allogeneic hematopoietic cell transplantation.

Statistical Analysis. We used two tailed t-tests and analysis ofvariance for the estimation of significant differences in CD47expression level across subgroups of AML based on morphologic,cytogenetic, and molecular categorizations. Associations between thehigh and low CD47 groups and baseline clinical, demographic, andmolecular features were analyzed using Fisher's exact and Mann-Whitneyrank sum tests for categorical and continuous variables, respectively.Two-sided p-values of less than 0.05 were considered to indicatestatistical significance.

The prognostic value of CD47 expression was measured through comparisonof the event-free and overall survival of patients with estimation ofsurvival curves by the Kaplan-Meier product limit method and thelog-rank test. Within this analysis, we first derived a binaryclassification of AML patients into High CD47 and Low CD47 expressiongroups by comparing the expression of CD47 (as measured by 213857_s_atwithin GSE1159) relative to an optimal threshold. This threshold wasdetermined using X-Tile16, a method which we employed to maximize thechi-square statistic between the two groups for the expected versusobserved number of deaths. This stratification segregates the 261 AMLpatients with available outcome data into two unequally sized groups,with 72% of patients with lowest expression considered CD47 low, and 28%with highest expression considered CD47 high. These two groups havedifferent overall survival with a hazard ratio of 1.42 for the CD47 highgroup, and a corresponding uncorrected p-value of 0.033, which requirescross-validation to avoid the risk of overfitting.

Accordingly, we assessed the validity and robustness of riskstratification using CD47 expression by applying this optimal thresholdto an independent test cohort of 242 NK-AML patients described byMetzeler et al. Notably, despite the presence of other variablespotentially confounding associations with survival (including moreadvanced age, and differing therapies), derivation of an optimalcutpoint using the 242 NK-AML patients within the test dataset yielded asimilar stratification, with 74% of patients with lowest expressionconsidered CD47 low, and 26% with highest expression considered CD47high.

Next, we assessed the validity of this stratification in across-validation cohort of 137 uniformly treated NK-AML patientsdescribed by Bullinger et al. Within this validation dataset, we couldsimilarly define two groups of similar size (i.e., 72% and 28% withlowest and highest CD47 levels, respectively), and these two groups hadsignificantly different outcomes when assessed for overall survival(FIG. 22B, p=0.002, hazard ratio 2.02, 95% CI 1.37 to 4.03), andevent-free survival (FIG. 223A, p=0.004, hazard ratio 1.94, 95% CI 1.30to 3.77). Of the 137 patients, 5 did not have reliable measurements forCD47 when using the data selection and normalization criteria describedby the authors.

To determine the robustness of this association, we also examined thepredictive value of CD47 expression when the validation cohort wasdivided into low and high CD47 expression groups based on expressionrelative to the median, or as a continuous variable. As above, higherCD47 expression was associated with worse event-free and overallsurvival. Of the 137 patients studied, a subset of 123 patients hadavailable survival data, CD47 expression data, and FLT3-ITD statusreported. Within this cohort, we assessed the relationship of CD47expression level as a continuous variable with outcome using univariateCox proportional-hazards analysis, with event-free survival or overallsurvival as the dependent variable. We used multivariateCox-proportional hazards analysis with event-free survival or overallsurvival as the dependent variable and FLT3-ITD status, age, andcontinuous expression level of CD47 as directly assessed independentvariables.

Associations of CD47 with other covariates (eg, NPM1, CEBPA) werelimited by sample size and missing data for covariates. The Wald testwas used to assess the significance of each covariate in multivariateanalyses. Univariate and multivariate proportional-hazards analyses weredone using the coxph function in the R statistical package.

Example 5 CD47 is a Prognostic Factor and Therapeutic Antibody Target onSolid Tumor Cancer Stem Cells

We have found that increased CD47 expression is associated with worseclinical outcomes in diffuse large B-cell lymphoma (DLBCL) and ovariancarcinoma (FIG. 24). Additionally, we have now found that anti-CD47antibodies enable the phagocytosis of cancer stem cells from bladdercancer, ovarian carcinoma, and medulloblastoma in vitro with humanmacrophages (FIG. 25).

Example 6 Therapeutic Antibody Targeting of CD47 Eliminates Human AcuteLymphoblastic Leukemia

Although standard multi-agent chemotherapy cures a significant number ofpatients with standard-risk pediatric ALL, these same therapies aresignificantly less effective in both the high-risk pediatric populationand in all adults with ALL. Thus, additional therapies are necessary tomore effectively treat these patient subsets. As an alternative tochemotherapy, monoclonal antibodies have recently emerged as anattractive therapeutic modality due to the ability to selectively targetleukemia cells, thereby minimizing systemic toxicity. Indeed, severalmonoclonal antibodies are currently in clinical trials for the treatmentof ALL.

CD47 is identified herein as a therapeutic antibody target in acutemyeloid leukemia (AML). It is investigated whether a blocking monoclonalantibody against CD47 could eliminate primary human ALL in vitro and invivo, in order to determine the use of an anti-CD47 antibody as atherapy in standard and high-risk ALL.

Materials and Methods

Human Samples.

Normal human bone marrow cells were purchased from AllCells Inc.(Emeryville, Calif., USA). Human ALL samples were obtained from patientsat the Stanford University Medical Center, with informed consent,according to an IRB-approved protocol.

Flow Cytometry Analysis.

The following antibodies were used for analysis of ALL and NBM cells:CD3 APC-Cy7 and CD19 APC (BD Biosciences, San Jose, Calif., USA).Analysis of CD47 expression was performed with an anti-human CD47 FITCantibody (clone B6H12.2, BD Biosciences). For human engraftment analysisin mice, the following antibodies were used: mouse Ter119 PeCy5, mouseCD45.1 PeCy7, human CD45 PB, human CD19 APC, and human CD3 APC-Cy7(Ebiosciences, San Diego, Calif., USA).

ALL Microarray Gene Expression Data and Statistical Analysis.

We used previously described methods for the univariate and multivariatestatistical analyses of CD47 gene expression data and its relationshipto clinical and pathological variables. Briefly, gene expression andclinical data were analyzed for three previously described cohorts ofALL patients: 1) a dataset of 360 pediatric ALL patients with B- andT-ALL subtypes, diverse risk profiles and corresponding therapiesincluding a subset (n=205) with available data on disease free survival.Microarray and clinical data were obtained from St. Jude Children'sResearch Hospital; 2) a dataset of 207 pediatric B-precursor ALLpatients with high-risk features uniformly treated through theChildren's Oncoloy Group Clinical Trial P9906 obtained from NCBI throughthe Gene Expression Omnibus (GSE11877); and 3) 254 pediatric ALLpatients registered to Pediatric Oncology Group trials stratified forthe presence of recurrent cytogenetic abnormalities and remission versusfailure within each cytogenetic group with data obtained from theNational Cancer Institute caArray. Affymetrix probeset summaries werederived from the corresponding microarray raw CEL data files using aCustom Chip Definition File derived from NCBI Reference Sequences(version 12), and then normalized using MAS 5.0 using BioConductor. Forsurvival analyses, NM 198793 at was selected as the probeset torepresent CD47 mRNA based on it demonstrating highest expression amongthe 3 probesets for CD47 on the microarrays, and based on its exonicstructure capturing the CD47 splice variant expressed in hematopoietictissues.

We assessed the relationship of CD47 mRNA expression and outcomes ascontinuous variables using univariate Cox proportional-hazards analysis,with disease free or overall survival as the dependent variable. Usingthe coxph function in the R statistical package, the Wald test was usedto assess the significance of each covariate, represented by the base-2logarithms of CD47 mRNA expression. For dichotomous stratification ofCD47 expression, we used maximally selected chi-square statistics asimplemented within X-tile to define an optimal threshold. To guardagainst erroneous overestimation of p-values through multiple hypothesistesting, we corrected the log-rank Kaplan-Meier p-values using theMiller-Siegmund method well as sub-sampling (n=1000) based internalcross-validation.

Therapeutic Antibodies.

Anti-human CD47 antibodies (B6H12.2, BRIC126, 2D3), anti-SIRPα antibody,IgG control, and anti-CD45 antibodies were used as described in theprevious examples. The anti-CD47 antibody clone BRIC126 was obtainedfrom AbD Serotec (Raleigh, N.C., USA).

Generation of Mouse and Human Macrophages.

Isolation of mouse and human macrophages were performed as previouslydescribed. Briefly, femurs and tibias from wild-type Balb/C mice wereharvested into a single cell suspension and incubated for 7-10 days inIMDM 10% fetal calf serum with 10 ng/ml murine M-CSF (Peprotech, RockyHill, N.J., USA) at 37° C. Cells were then washed and adherent cellstrypsinized and plated for in vitro phagocytosis assays. For humanmacrophages, mononuclear cells were isolated from human peripheral bloodby ficoll density gradient centrifugation and plated onto 10 cm petridishes at 37° C. for one hour. Non-adherent cells were then washed offand the remaining adherent cells were incubated for 7-10 days in IMDMwith 10% human AB serum. Cells were then trypsinized and plated for invitro phagocytosis assays.

In Vitro Phagocytosis Assays.

Phagocytosis assays were performed as described in the previousexamples. Briefly, bulk ALL cells were CFSE-labeled and incubated witheither mouse or human macrophages in the presence of 10 μg/ml of theindicated antibodies at a target:effector cell ratio of 4:1(2×10⁵:5×10⁴). Incubation occurred at 37° C. for 2 hours and thenanalyzed by fluorescent microscopy for phagocytosis using the phagocyticindex: number of cells ingested per 100 macrophages.

Ex Vivo Antibody Coating of ALL Cells.

Human ALL cells were incubated with 30 μg/ml of either IgG1 isotypecontrol, anti-CD45, or anti-CD47 antibody for 30 minutes at 4° C. Cellswere washed and then 1-4×10⁶ cells were transplanted intosublethally-irradiated NOD.Cg-PrkdcscidIl2rgtmlWjl/SzJ (NSG) adults orpups and analyzed for ALL engraftment in the peripheral blood and bonemarrow 6-10 weeks later. Antibody coating of ALL cells was confirmed byflow cytometry with a secondary antibody prior to transplantation intomice. Sublethal irradiation was 230 rads and 100 rads for NSG adults andpups, respectively.

In Vivo Treatment of Human ALL Engrafted Mice.

1-4×10⁶ bulk human ALL cells were transplanted intravenously via theretro-orbital sinus into sublethally-irradiated (230 rads) adult NSGmice. Alternatively, ALL cells were transplanted into the facial vein of2-4 day old sublethally-irradiated (100 rads) NSG pups. Six to ten weekslater peripheral blood and bone marrow ALL engraftment (B-ALL:hCD45+CD19+; T-ALL: hCD45+CD3+) was assessed by tail bleed andaspiration of the femur, respectively. Engrafted mice were treated for14 days with daily 100 μs intraperitoneal injections of either IgGcontrol or anti-CD47 antibody (clone B6H12.2). On day 15, mice weresacrificed and analyzed for ALL engraftment in the peripheral blood,bone marrow, spleen, and liver.

Bone Marrow Tissue Section Preparation and Staining.

Mouse tibias from antibody-treated NSG mice were harvested and preservedin formalin. Hematoxylin and eosin staining and immunohistochemistry ofhuman CD45+ cells were performed by Comparative Biosciences Inc.(Sunnyvale, Calif., USA).

Results

CD47 Expression is Increased on a Subset of Human ALL Cells Compared toNormal Bone Marrow.

To determine whether CD47 may be involved in the pathogenesis of ALL, wefirst investigated CD47 cell surface expression on primary human ALL andnormal bone marrow cells by flow cytometry. We surveyed 17 diversepatients with ALL that included both precursor B and T lineage subtypes.Compared to normal mononuclear bone marrow cells, CD47 was more highlyexpressed on human ALL samples, approximately 2-fold when consideringall samples, with similar expression between B and T subtypes. However,assessing CD47 mRNA expression in a large cohort of ALL patients, wefound that T-ALL patients expressed significantly higher levels comparedto B-ALL patients.

CD47 Expression is an Independent Prognostic Predictor in Mixed andHigh-Risk ALL

Since CD47 expression was increased on ALL samples, and given theobserved heterogeneity in CD47 expression across ALL subtypes, weinvestigated whether the level of CD47 expression correlated withclinical prognosis. First, CD47 expression was investigated as aprognostic predictor in pediatric ALL patients with mixed risk andtreatment utilizing gene expression data from a previously describedpatient cohort. 360 patients were stratified into high and low CD47−expressing groups based on an optimal cutpoint and clinical outcomeswere determined. Among the subset of this cohort with available outcomedata (n=205), patients expressing higher levels of CD47 had worseoutcomes, whether CD47 expression was tested as a continuous variable(p=0.03; HR 1.78 per 2-fold change in CD47 expression; 95% CI1.05-3.03), or as a dichotomous variable relative to an internallyvalidated optimal threshold (uncorrected p=0.0005, correctedp=0.01; HR3.05; 95% CI 1.49-6.26) (FIG. 26A).

Second, to investigate the prognostic power of CD47 expression inhigh-risk ALL patients, clinical outcome in a uniformly treatedpreviously described cohort of 207 high-risk pediatric ALL patients wasinvestigated. For this cohort, high-risk was defined by age>10 years,presenting WBC count>50,000/μl, hypodiploidy, BCR-ABL positive disease,and central nervous system or testicular involvement. In these high-riskALL patients, higher CD47 expression correlated with a worse overallsurvival when CD47 expression was again considered as either acontinuous variable (p=0.0009, HR 3.59 per 2-fold change in CD47expression; 95% CI 1.70 to 7.61), or as a dichotomous one relative to aninternally validated optimal threshold (uncorrected p=0.001, correctedp=0.01; HR 2.80; 95% CI 1.21 to 6.50) (FIG. 26B). In multivariateanalysis, CD47 expression remained a significant prognostic factor whenage at diagnosis, gender, WBC count, CNS involvement, and minimalresidual disease were considered as covariates (FIG. 26C).

Lastly, we utilized a third independent gene expression dataset toinvestigate whether CD47 expression could predict disease relapse.Indeed, CD47 expression was higher in patients failing to achieve acomplete remission (CR) compared to those that did achieve a CR (FIG.26D). Taken together, these separate observations among distinct anddiverse cohorts establish that higher expression of CD47 is anindependent predictor of adverse outcomes in pediatric patients withstandard- and high-risk ALL, including induction failure, relapse, anddeath.

Blocking Monoclonal Antibodies Against CD47 Enable Phagocytosis of ALLCells.

Next, we investigated whether ALL cells could be eliminated bymacrophage phagocytosis enabled through blockade of the CD47-SIRPαinteraction with a blocking anti-CD47 antibody. First, we incubatedhuman macrophages with fluorescently-labeled ALL cells in the presenceof an IgG1 isotype control, anti-CD45 isotype-matched, or anti-CD47antibody and measured phagocytosis by immunofluorescence microscopy. Twodifferent blocking anti-CD47 antibodies (B6H12.2 and BRIC126) enabledphagocytosis of ALL cells compared to IgG1 isotype and anti-CD45 controlantibodies as measured by significant increases in the phagocytic index.In addition, anti-CD47 antibodies enabled phagocytosis of all ALLsubtypes profiled, including those with cytogenetically high-risk(Ph+ALL and MLL+ALL). Since several studies report that CD47-SIRPαsignaling may be species-specific, the ability of anti-CD47antibody-coated human cells to be phagocytosed by mouse macrophages wasdetermined before proceeding with in vivo antibody treatment experimentsin mouse xenotransplants. Similar to human macrophages, two blockinganti-CD47 antibodies (B6H12.2 and BRIC126) enabled phagocytosis of ALLcells by mouse macrophage effectors compared to IgG1 isotype andanti-CD45 antibody controls. Furthermore, no phagocytosis was observedwith a non-blocking anti-CD47 antibody (2D3). Lastly, blockade of SIRPαwith an anti-mouse SIRPα antibody also resulted in increasedphagocytosis, thus supporting the mechanism of increased phagocytosisresulting from disruption of the CD47-SIRPα interaction.

Ex Vivo Coating of ALL Cells with an Anti-CD47 Antibody InhibitsLeukemic Engraftment.

The ability of a blocking anti-CD47 antibody to eliminate ALL in vivowas investigated by two independent methods. First, the ability ofanti-CD47 antibody to inhibit ALL engraftment was determined using anantibody pre-coating assay. ALL cells were coated ex vivo with eitherIgG1 isotype control, anti-CD45, or anti-CD47 antibody (B6H12.2),transplanted into sublethally-irradiated immunodeficient NOD/SCID/Il2γrnull (NSG) mice, and measured for ALL engraftment in the peripheralblood and bone marrow 6-10 weeks later. Prior to transplantation,coating of ALL cells with antibody was verified by flow cytometry (FIG.27A). Antibody pre-coating experiments were performed with both primaryB- and T-ALL samples to include the two major disease subtypes.Anti-CD47 antibody significantly inhibited leukemic engraftment of bothB- and T-ALL cells in the peripheral blood (FIG. 27B) and bone marrow(FIG. 27C) compared to IgG1 isotype or anti-CD45 antibody controls.Pre-coating with the anti-CD47 antibody nearly completely eliminated ALLengraftment in vivo.

Anti-CD47 Antibody Eliminates ALL Engraftment in the Peripheral Bloodand Bone Marrow.

In the second method of investigating the in vivo efficacy of ananti-CD47 antibody against human ALL, mice were first stably engraftedwith ALL cells and then treated with antibody. 1-4×10⁶ B- or T-ALL cellswere transplanted into sublethally-irradiated NSG newborn pups oradults. Six to ten weeks later, ALL engraftment was measured in theperipheral blood and bone marrow by flow cytometry. Those mice that hadsignificant levels of ALL engraftment were then selected for in vivoantibody therapy (FIG. 28A), as determined by greater than 10% humanchimerism in the peripheral blood and/or bone marrow with engraftmentranging from 10-97%. ALL engrafted mice were treated with dailyintraperitoneal injections of 100 μg IgG control or anti-CD47 antibody(B6H12.2) for 14 days. This dosing regimen was selected based on ourprior study demonstrating elimination of AML in mouse xenotransplants.Tumor burden was then measured post-treatment in the peripheral bloodand bone marrow by flow cytometry. Compared to IgG control, anti-CD47antibody therapy reduced the level of circulating leukemia, and in mostcases eliminated ALL from the peripheral blood (FIG. 28 A,B). Thiseffect was observed for mice transplanted with both B- and T-ALL cells.Similarly, anti-CD47 antibody reduced or eliminated ALL engraftment inthe bone marrow, while ALL disease burden increased with IgG controltreatment (FIG. 28C). Bone marrow histology of antibody-treated micerevealed infiltration of monomorphic leukemic blasts in controlIgG-treated mice (FIG. 28D). Anti-CD47 antibody-treated bone marrowdemonstrated normal mouse hematopoietic cells with cleared hypocellularareas. Immunohistochemistry of mouse marrows confirmed near completeinvasion of human CD45-positive leukemic blasts in IgG-treated marrowcompared to few human CD45-positive leukemia cells observed in anti-CD47antibody-treated marrow (FIG. 28D).

Anti-CD47 Antibody Eliminates ALL Engraftment in the Spleen and Liver.

Hepatomegaly and splenomegaly can cause clinical complications and are acommon finding in ALL, being observed in up to 69% of patients atdiagnosis. To determine whether anti-CD47 antibody could potentiallytreat hepatosplenomegaly in ALL, we investigated the ability ofanti-CD47 antibody to eliminate ALL engrafted in the spleen and liver.Of the ALL samples utilized for in vivo treatment studies, we identifiedthree B-ALL patient samples (ALL8, ALL21, and ALL22) that gave rise todisease in the spleen and/or liver, with associated splenomegaly, whentransplanted into NSG mice. These mice were treated for 14 days with theidentical regimen of either IgG or anti-CD47 antibody as in FIG. 28 withspleen weights and ALL chimerism in the spleen and liver measuredpost-treatment. Control IgG-treated B-ALL-engrafted mice exhibitedsignificant splenomegaly compared to untransplanted NSG mice (FIG.29A,B). In contrast, anti-CD47 antibody treatment reduced ALL-inducedsplenomegaly to spleen sizes similar to untransplanted NSG mice (FIGS.29A,B). To determine whether this effect was due to direct eliminationof ALL cells in the spleen, the spleens of B-ALL-engrafted mice treatedwith IgG control or anti-CD47 antibody were analyzed for ALL diseaseburden. Compared to IgG-treated mice, anti-CD47 antibody significantlyeliminated B-ALL engraftment in the spleen (FIG. 29C). Similarly,anti-CD47 antibody significantly eliminated ALL in the liver compared tothe extensive leukemic infiltration observed with control IgG treatment(FIG. 29D). These results indicate that anti-CD47 antibody is highlyeffective in eliminating ALL in the spleen and liver, in addition to theperipheral blood and bone marrow.

We report here that CD47 is expressed at high levels on a large subsetof human ALL subtypes, that cell surface CD47 is a monoclonal antibodytarget for eliminating ALL blasts by enhancing innate immune systemrecognition of leukemic blasts by macrophage-mediated phagocytosis, andthat CD47 itself is an independent prognostic variable in ALL that canpredict disease free survival, overall survival, and relapse in bothmixed and high-risk ALL patients. Together, these data show that ALLpathogenesis relies on mechanisms to evade innate immune recognition andthat modulation of the innate immune recognition of tumor cells is aviable treatment modality.

Within the last few years, several cell surface proteins have beenidentified as candidate targets, and some monoclonal antibodies haveproceeded into early and late phase clinical trials. Most therapeuticantibodies in clinical development have been focused on B-ALL. One suchcandidate is CD20, as its expression is observed in approximately 40 to50% of B-ALL cases. Rituximab, an anti-CD20 antibody, initially approvedfor treatment of B cell lymphoma, has demonstrated a significantsurvival advantage when added to standard chemotherapy in some ALLclinical trials, particularly the Burkitt's subtype. Although effectivein adult CD20+ B-ALL, there is a paucity of clinical data on theefficacy of rituximab in pediatric ALL. In contrast to CD20, CD22 isexpressed in a larger percentage of B-ALL cases and is present ongreater than 90% of B-ALL patients. Epratuzumab, a humanized monoclonalanti-CD22 antibody, is currently being investigated in clinical trials.Although early clinical studies with epratuzumab as a single agent inrelapsed ALL showed limited effect, anti-CD22 antibody-immunotoxinconjugates may improve the efficacy of epratuzumab, since CD22 isreported to be rapidly internalized upon antibody binding. Severalimmunoconjugates directed against CD22 are currently being explored inPhase I trials. In addition, antibodies and immunotoxins to otherantigens including CD19 are currently being explored.

Although several therapeutic antibodies are in clinical development forB-ALL, there are relatively few antibody therapies for treatment ofT-ALL. The most prominent antibody for T-ALL, alemtuzumab, is targetedat CD52, as it is expressed on greater than 95% of normal lymphocytesand at higher levels in T compared to B lymphoblasts. Althoughpre-clinical data suggest potential efficacy of alemtuzumab, early phaseclinical trials do not report a significant benefit as a single agent orin combination with chemotherapy for the treatment of relapsed T-ALL.

In contrast to the targeted therapies developed for B-ALL and T-ALL, ourdata strongly suggest that an anti-CD47 antibody can be effective ineliminating both B- and T-ALL and thus could increase the number oftherapeutic options for both. Because anti-CD47 antibody treatment mayeliminate ALL blasts with limited toxicity and is equally effective intargeting low, standard, and high-risk ALL, these results provide astrong rationale for development of an anti-CD47 antibody for thetreatment of ALL patients.

Example 7 Expression of CD47 on Solid Tumor Cells and Manipulation ofPhagocytosis of the Same

Several anti-human CD47 monoclonal antibodies have been generated,including some capable of blocking the CD47-SIRP□ interaction (B6H12 andBric126) and others unable to do so (2D3). We tested the ability ofthese antibodies to enable phagocytosis of ovarian, bladder, and coloncancer cells by macrophages in vitro, and to alter the survival ofanimals engrafted with these cancer cells in vivo. In contrast to cellstreated with an IgG1 isotype control or non-blocking anti-CD47 antibody(2D3), tumor cells treated with blocking anti-CD47 antibodies B6H12 orBric126 were efficiently phagocytosed by mouse and human macrophages.Colon cancer stem cells (Linneg, EpCAM+, CD44+, CD166+) were isolated byFluorescence Activated Cell Sorting (FACS) from patient tumor samples.

The cell samples tested were as follows:

Ovarian. Patient ovarian cancer (OC) cells were engrafted into theperitoneal cavity of NSG mice. Prior transduction of these cells with alentivirus designed to express GFP and luciferase enabled the use ofbioluminescent imaging to monitor tumor growth. After confirmingengraftment of the OC cells, mice were treated daily with anintraperitoneal injection of 400 □g anti-CD47 (clone Bric126) or controlmouse IgG. Tumor growth was then evaluated biweekly with bioluminescentimaging.

The fold change in total flux (photons/sec) is shown for each mouseafter each individual mouse was normalized to its respectivepretreatment value. IgG treated mice (n=9) are represented by redcircles. Anti-CD47 treated mice (n=10) are represented by bluetriangles. The horizontal line represents the mean bioluminescent signal(plus standard error) of each treatment group. The fold differencebetween the two treatment groups is shown at each time point.

Pancreatic. Pancreatic cancer (PANC1) cells were transduced with alentivirus designed to express GFP and Luciferase. Successfullytransduced (GFP+) cells were isolated by FACS. 500,000 transduced PANC1cells were directly injected into the pancreas of NSG mice. After sevendays, engraftment of PANC1 cells was quantified by bioluminescentimaging. Mice were then treated daily with 500 □g control IgG (n=5) oranti-CD47, clone B6H12 (n=5). Tumor growth was monitored and quantifiedweekly by bioluminescent imaging. Each symbol represents an individualmouse.

Breast. 10⁶ cells from a patient breast cancer xenograft were engraftedinto the mammary fat pad of NSG mice. Daily intraperitoneal injectionsof either 400 □g control IgG or anti-CD47 (B6H12) were initiated 2 weeksafter cells were injected. Antibody treatment was stopped after 8 weeks.Tumor formation occurred in all control IgG treated mice. Importantly,the anti-CD47 treated mice were evaluated 3 months after stoppingantibody treatment, and still no tumor formation was detected in any ofthe mice, indicating that the anti-CD47 antibody successfully targetedand eliminated the breast CSCs. Representative images of the mammary fatpads of 3 mice from each treatment group

Colon. Patient colon cancer cells were engrafted subcutaneously on theback of of NSG mice. Prior transduction of all injected cells with alentivirus designed to express GFP and luciferase enabled the use ofbioluminescent imaging to monitor tumor growth. After confirmingengraftment of the colon cancer cells, mice were treated daily with anintraperitoneal injection of 500 □g anti-CD47 (clone B6H12), anti CD44(Hermes-3), control mouse IgG, or a combination of anti-CD44 andanti-CD47 antibodies. Tumor growth was then evaluated weekly withbioluminescent imaging.

Bladder Metastasis. Tumor cells from a patient bladder cancer samplewhich reliably forms metastases to they lymph nodes and lungs wereinjected subcutaneously onto the back of NSG mice. Treatment withanti-CD47 (400 □g/day), anti-CD44 (150 □g, MWF), or Herceptin (200□g/week) antibodies was initiated upon detection of a palpable tumormass. The number of metastases was determined by gross examination ofexcised lymph nodes at the conclusion of the experiment. The number ofmicrometastases was determined by a trained pathologist on sections cutfrom lungs preserved in 10% buffered formalin phosphate.

Expression of CD47 on various cancer cells is shown in Table 3:

Flow Cytometry Of Immunoflouresence Dissociated Cancer Tissue StainingOn Frozen Percent CD47 Positive Tissue CD47 Expression Tumor Type TumorCells On Cancer Cells Ovarian 58-95 Positive Breast 89 Not DeterminedColon 73-97 Not Determined Bladder 84-98 Positive Head & Neck 19-86 NotDetermined Lung Not Determined Positive Melanoma 98 PositiveGlioblastoma 20-97 Not Determined CD47 is expressed on human tumorcells. CD47 was evaluated by flow cytometry (middle column) ondissociated patient primary or xenograft tumors from various tissues.“Tumor cells” are defined as live, lineage negative cells, where lineagerepresents human CD45 negative CD31 negative (primary samples) or mouseCD45 negative, H-2K^(d/b) negative (xenograft samples) cells.Immunoflouresence staining (right column) was performed on sections cutfrom a subset of primary and xenograft tumor samples preserved in OCTimmediately upon collection. Where indicated, CD47 expression wasobserved on bulk cancer cells.

As shown in FIG. 30, antibodies targeted to human CD47 enable thephagocytosis of OC cells. A: CFSE-labeled primary human bladder cancercells were incubated with human macrophages in the presence of theindicated antibodies and assessed for the presence of tumor cells withinmacrophages. B-C: Phagocytosis of patient ovarian cancer cells (B) orcolon cancer stem cells (C) resulting from indicated antibody treatmentwas quantified. Each dot color represents a different primary tumorsample. The phagocytic index was determined as the number of OC cellspresent within 100 macrophages.

As shown in FIG. 31, antibodies targeted to CD47 inhibit the growth ofpatient tumors. A-D: Tumor cells from ovarian (A), pancreatic (B),breast (C), or colon (D) tumors were engrafted into immunodeficientmice. These mice were then treated with control IgG or anti-CD47antibodies and tumor growth was assessed directly or by bioluminescence.In all cases, anti-CD47 antibody treatment substantially inhibited tumorgrowth. E-F: Tumor cells from patient bladder were injectedsubcutaneously into immunodeficient mice and treated with the indicatedantibodies. Anti-CD47 antibody treatment significantly inhibitedmetastasis to the lymph nodes (E) and formation of micrometastases inthe lungs (F). The total number of metastases detected in each treatmentgroup is indicated.

Example 8 Expression of CD47 in Brain Tumor

Acquisition of Brain Tumor Samples: Freshly resected brain tumor sampleswere obtained from the department of Neurosurgery under IRB approvedprotocols. Samples are minced using a sterile scalpel and washed in HBSSto remove debris. Minced tissue is then incubated in collagenase IV(img/ml) for 60-90 minutes at 37° C. with constant agitation.Dissociated cells are then passed sequentially through a 100, 70 and 40□m cell strainer and washed in HBSS. Dead cells are removed by densitycentrifugation in 0.9M sucrose and then treated with ACK-RBC lysisbuffer (invitrogen) to remove red blood cells. Cells are then collectedby centrifugation and resuspended in FACS buffer.

FACS Staining: Single cell suspension were stained with CD133/1-APC andCD133/2-APC (Miltenyi) and CD47-PE (BD biosciences) and analyzed onARIA-II (BD Biosciences).

Results:

In 10 gliomas analyzed, 10/10 tumors were CD47⁺ with varying degree ofCD47 expression. An average of 66% of the cells were CD47⁺, where asonly 4/10 tumors were CD133⁺. In the CD133⁺ tumors all CD133⁺ cells alsoexpressed CD47.

This data suggests that anti-CD47 therapy can be a viable avenue ofinvestigation in human glioblastoma. This also suggests that at least inCD133⁺ tumors targeting CD47 would also target the cancer stem cellpopulation.

Tumor % of the viable, CD45-cells: Date CD133+ CD47+ CD47+133+ (%) Nov.15, 2006  45% 95%  53% Jun. 29, 2007   0% 97.1%   0 Oct. 1, 2007   0%46% 0 Feb. 12, 2008   0% 20% 0 Mar. 27, 2008   0% 31% 0 Apr. 10, 20087.6% 68% 8.5% Apr. 28, 2008 9.1% 74% 8.8% Jun. 2, 2008 4.5% 92% 8.5%Jul. 15, 2008 0.0% 37% 0.0% Oct. 9, 2008 0.0% 95% 0.0%

An analysis of survival vs. gene expression data for cd47 andCD133/Prom1 shows that patients with glioblastoma whose tumors expresslow CD47 have better survival (p=0.0239).

What is claimed is:
 1. A method of treating a human subject for ovariancancer, the method comprising: administering to a human subject in needthereof an antibody that disrupts the binding of CD47 with SIRPα, at adose that achieves a depletion in ovarian cancer cells by increasingphagocytosis of cancer cells.
 2. The method of claim 1, wherein theantibody that disrupts the binding of CD47 with SIRPα specifically bindsto CD47.
 3. The method of claim 1, wherein phagocytosis of the cancercells is increased with no significant increase in phagocytosis ofnormal CD34+ cells.
 4. The method of claim 1, wherein the antibody is abispecific antibody, or is administered with a second monoclonalantibody directed against a specific cancer cell marker for depletion ofcells expressing the marker.
 5. The method of claim 1, wherein theantibody is characterized by up-regulating phagocytosis of cancer cellsin an in vitro assay with human macrophages by at least 10% compared tophagocytosis in absence of the antibody.
 6. The method of claim 5,wherein the antibody is characterized by up-regulating phagocytosis ofcancer cells in an in vitro assay with human macrophages by at least 20%compared to phagocytosis in absence of the antibody.
 7. The method ofclaim 6, wherein the antibody is characterized by up-regulatingphagocytosis of cancer cells in an in vitro assay with human macrophagesby at least 50% compared to phagocytosis in absence of the antibody. 8.A pharmaceutical composition comprising: a dose of an antibody thatdisrupts the binding of CD47 with SIRPα, in an amount that is effectiveto deplete ovarian cancer cells by increasing phagocytosis of the cancercells in a human subject upon administration.
 9. The composition ofclaim 8, wherein the antibody specifically binds to CD47.
 10. Thecomposition of claim 8, wherein the phagocytosis of cancer cells isincreased with no significant increase in phagocytosis of normal CD34+cells.
 11. The composition of claim 8, wherein the antibody ischaracterized by up-regulating phagocytosis of cancer cells in an invitro assay with human macrophages by at least 10% compared tophagocytosis in absence of the antibody.
 12. The composition of claim11, wherein the antibody is characterized by up-regulating phagocytosisof cancer cells in an in vitro assay with human macrophages by at least20% compared to phagocytosis in absence of the antibody.
 13. Thecomposition of claim 12, wherein the antibody is characterized byup-regulating phagocytosis of cancer cells in an in vitro assay withhuman macrophages by at least 50% compared to phagocytosis in absence ofthe antibody.